Method and compositions for evaluating risk of developing type 2 diabetes in people of chinese descent

ABSTRACT

Methods and compositions for identifying mutations and polymorphisms in mutant genes encoding gene product involved in insulin secretion, for example, hepatocyte nuclear factor-1∝, glucokinase, amylin and mitochondrial DNA are disclosed. Specifically, a microchip comprising a combination of at least two different mutant genes wherein each gene comprises at least one mutation indicative of a predisposition for type-2 diabetes in a member of a Chinese population is disclosed. A kit comprising the microchip, an isolated nucleic acid, primers and probes which are specifically used to screen or identify the mutations in genes of hepatocyte nuclear factor-1∝, glucokinase, amylin and mitochondrial DNA are also disclosed.

FIELD OF THE INVENTION

This subject invention relates to the identification and use ofmutations and polymorphisms in mutant genes of wild-type genes involvedin insulin secretory function that are associated with the increasedrisk of a Chinese individual to develop type 2 diabetes. The inventionis exemplified by a combination of mutations, uniquely identified inChinese individuals with a positive family history of type 2 diabetes,in the genes encoding hepatocyte nuclear factor-1∝, glucokinase, amylinand mitochondrial DNA. The combination of mutated genes finds use inscreening Chinese individuals at risk of developing type 2 diabetes andin providing physicians with information to enable them to apply patienttailored therapies.

BACKGROUND

Although people of Chinese ancestry account for >20% of the world'spopulation (Chan, et al(1997) 20: 1785), very little is known about thegenetic factors that contribute to the development of diabetes in thispopulation. The prevalence of diabetes amongst Chinese people variesfrom <1% in some rural areas in mainland China to 6-12% in Hong Kong,Singapore, and Taiwan (Chan, et al (1997), supra). Hong Kong can beregarded as a paradigm of future China.

The prevalence of diabetes mellitus is reaching epidemic proportionsamongst Hong Kong Chinese, with type 2 diabetes being the predominantform in pateints with early-or late-onset of disease (Chan and Cockram(1997) Diabetes Care 20: 1785). Type 2 diabetes mellitus is aheterogeneous disease that is caused by both genetic and environmentalfactors. The age-adjusted prevalence of diabetes in the Chinesepopulation has increased from 7.7% in 1990 (Cockram, et al (1993)Diabetes Res and Clin Practice 21: 67) to 8.9% in 1995 (Cockram and Chan(1999) In: Diabetes in the New Millennium, Pot Still Press, Sydney, pp.11-22). In a population-based study conducted in 1995, the crudeprevalence of diabetes mellitus was 9.6%, rising from 1.7% in those agedunder 40 years to 25% in those older than 60 years (Janus (1997) ClinExp Pharmacol Physiol 24: 987). There is a high prevalence of obesity(43%) and positive family history of diabetes (50%) in Chinese patientspresenting with acute or early onset diabetes (Chan, et al (1993)Postgrad Med J 69: 204; Ko, et al (1998) 35: 761). These findingsindicate that genetic factors, in addition to environmental factors, canbe an important cause of early onset diabetes in this population.

Because type 2 diabetes is an insidious disease, it is estimated that asmany as half of the individuals in Hong Kong that would be considereddiabetic remain undiagnosed. Most patients are finally diagnosed onlywhen presenting with overt symptoms that often are the consequence ofadvanced disease. Clinic as well as population-based studies reveal thatabout 17% of diabetic patients in Hong Kong are diagnosed before age 35years (Chan, et al (1993) Postgrad Med 69: 204; Janus (1996) The HongKong cardovascular risk factor prevalence study 1995-1996 Dept of ClinBiochem, Queen Mary Hospital of Hong Kong, Hong Kong, 1997). Due totheir anticipated long duration of disease, it is important to classifyand characterize the nature of diabetes in these young patients tofacilitate early diagnosis and appropriate treatments. Current methodsof diagnosing type 2 diabetes generally involve assessing phenotypicparameters, such as measuring fasting serum glucose levels byadministering an oral glucose tolerance test (OGTT) to determineimpaired glucose tolerance (IGT) or impaired fasting glucose (IFG).Phenotypic assessments of persons suspected of having type 2 diabetesare important, but they are limited in that patients generally receive adiagnosis only after presentation with overt symptoms. Furthermore,because the common symptoms of type 2 diabetes are a consequence of acombination heterogenous genetic and environmental causes, the therapiesprovided are general with regard to the disease rather than targetted tothe specific etiology of the individual patient. Numerous studies haveattempted to correlate the increased risk for development of type 2diabetes with a mutation of a specific gene, but the results of thesestudies repeatedly demonstrate that no one mutated gene can beattributed as the major cause of type 2 diabetes, emphasizing theheterogeneous nature of this disease. Furthermore, a mutation in aparticular gene that correlates with increased risk for developing type2 diabetes in individuals of one ethnic population is not relevant toindividuals of a second ethnic population, wherein the risk for type 2diabetes in individuals of the second ethnic population will correlatewith a different mutation or a mutation in a completely different gene.

It is therefore of interest to identify additional genetic mutations andpolymorphisms that are indicative of an increased risk for developingtype 2 diabetes in people of Chinese ancestry, and to develop methodsthat can be effectively employed to prophylactically identifyasymptomatic Chinese individuals with a genetic predisposition for type2 diabetes.

Relevant Literature

Maturity-onset diabetes of the young (MODY) is a monogenic form ofdiabetes characterized by autosomal dominant inheritance, early onset(usually before 25 years of age) and a primary defect in pancreaticβ-cell function (Fajans (1990) Diabetes Care 13: 49; Chan, et al (1990)Diabetic Med 7: 211; Byrne, et al (1996) Diabetes 45: 1503). This formof diabetes can result from mutations in at least five different genesincluding those encoding the glycolytic enzyme glucokinase (Froguel, etal (1993) New Engl J Med 328: 697), the liver-enriched transcriptionfactors expressed in the pancreatic β-cell, which are hepatocyte nuclearfactors HNF-1α (Yamagata, et al (1996) Nature 384: 455), HNF-1β(Horikawa, et al (1997) Nature Genet 17: 384), and HNF-4α (Yamagata, etal (1996) Nature 384: 458), and insulin promoter factor-1 (IPF-1)(Stoffers, et al (1997) Nature Genet 17: 138).

Some mutations and polymorphisms in the glucokinase and HNF-1α genesthat are associated with the genetic predisposition of a Chineseindividual to develop type 2 diabetes mellitus have been initiallyidentified in Ng, et al (Diabetic Medicine 1999, 16: 956, hereinincorporated by reference), but this manuscript does not disclose howthese mutations and polymorphisms might be used to identify Chineseindividuals with increased risk of developing type 2 diabetes.

U.S. Pat. No. 5,541,060 discloses the results of screening a cohort ofsixteen French families having MODY and the identification of severalmissense mutations in the glucokinase gene, however none of themutations identified are relevant to individuals of Chinese descent.U.S. Pat. No. 5,800,998 discloses a point mutation at nucleotide 414 ofhuman HNF 1α, but this single point mutation is not associated with agenetic predisposition of a Chinese individual to develop type 2diabetes.

Major susceptibility loci for non-insulin dependent diabetes have beenidentified through genome scans of individuals in Mexican-American(Hanis, et al (1996) Nature Genet 13: 161) and Finnish (Mahtani, et al(1996) Nature Genet 14: 90) populations, but not in individuals of aChinese population. Specific microsatellite regions of genomic DNA canbe correlated with major susceptibility loci that closely associate withthe increased risk of a Chinese subject to develop type 2 diabetes. Forinstance, Le Stunff, et al (Nature Genet. (2000) 26: 444) have reportedthat particular alleles of the insulin gene variable number of tandemrepeat (VNTR) locus are associated with obesity and type 2 diabetes.Also, microsatellite polymorphisms flanking the glucokinase have beenassociated with type 2 diabetes in a Taiwanese population (Wu, et al(1995) Diabetes Res Clin Pract 30: 21).

SUMMARY OF THE INVENTION

Compositions and methods are provided, wherein a unique combination ofgenetic markers indicative of a genetic predisposition for developingtype 2 diabetes in members of a Chinese population is described. Theinvention is exemplified by a combination of mutated gene sequences fromwild-type genes that are involved in insulin secretory function,including hepatocyte nuclear factor 1α (HNF-1α), glucokinase, amylin andmitochondrial DNA. The combination of representative mutations includeG20R, A116V, IVS2nt→GA, R203H, S432C and 1618M of HNF-1α; V101M, 1110T,A119D, Q239R and G385V of glucokinase; S20G of amylin; and A3243G ofmitochondrial tRNA^(Leu(UUR)). The combination of the mutated genes ofinterest will be most efficiently used for screening individuals atincreased risk by attaching them to a microchip.

Embodiments of methods for determining or detecting the geneticpredisposition of a Chinese individual to develop type 2 diabetesinclude obtaining a sample containing genomic nucleic acid from aChinese patient, such as a tissue biopsy or a blood sample, andcontacting that sample with a representative combination of at least twomutated genes of interest, then subjecting the sample DNA together withthe patient's DNA to hybridization conditions stringent enough to detectnucleotide differences of at least one base pair. Alternatively,particular genes of interest from the genomic DNA of a Chineseindividual at risk are screened using PCR primer pairs and PCR-RFLPtechniques to identify the presence or absence of a mutation known to beassociated with type 2 diabetes. The methods further encompass screeningthe genomic DNA of Chinese individuals who have been diagnosed with type2 diabetes or who have a primary family member with type 2 diabetes foradditional associative mutations in identified genes or for mutationscorrelative with the predisposition of a member of a Chinese populationto develop type 2 diabetes in additional candidate genes, such as thoseassociated with diabetic kidney disease and obesity.

The invention further provides for nucleic acid primers and probes thatare specifically used to identify mutations, for instance by PCR orhybridization, of wild-type genes involved in insulin secretion that areassociated with an increased risk of a Chinese subject to develop type 2diabetes. Additionally, proteins translated from genes carrying at leastone mutation associated with increased risk of a Chinese individual todevelop type 2 diabetes find use in functional diagnostic assays and inthe production of diagnostic antibodies that bind to the mutant but notthe wild-type protein.

The prophylactic detection of mutations and polymorphisms that areindicative of a genetic predisposition of a Chinese individual todevelop type 2 diabetes finds application in providing clinicians withinformation that allows for early detection and therapy initiationbefore the onset of overt symptoms or complications, and that enablesclinicians to administer specifically targetted therapies that addressthe etiology of an individual's disease.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows the nucleic acid sequence of human nuclear factor 1a(HNF-1α) exon 1 with the G20R mutation (SEQ ID NO: 1). The wild-typesequence is GenBank number U72612. FIG. 1B shows the nucleic acidsequence HNF-1α exon 2 with the A116V mutation (SEQ ID NO: 2). Thewild-type sequence is GenBank number U72613.

FIG. 2 shows the nucleic acid sequence of HNF-1α exons 3 and 4 depictingthe splice acceptor site mutation IVS2nt-1G→A (SEQ ID NO: 3) and themissense mutation R203H (SEQ ID NO: 4). The wild-type sequence isGenBank number U72614.

FIG. 3A shows the nucleic acid sequence of HNF-1α exons 5 and 6 with theS432C mutation (SEQ ID NO : 5). The wild-type sequence is GenBank numberU72615. FIG. 3B shows the nucleic acid sequence of HNF-1α exon 10 withthe I618M mutation (SEQ ID NO: 6). The wild-type sequence is GenBanknumber U72618.

FIG. 4A shows the nucleic acid sequence of human glucokinase exon 3,depicting the mutations V101M (SEQ ID NO: 7), I110T (SEQ ID NO: 8) andA119D (SEQ ID NO: 9). The wild-type sequence is GenBank number AF041016.FIG. 4B shows the nucleic acid sequence of human glucokinase exon 7 withthe Q239R mutation (SEQ ID NO: 10). The wild-type sequence is GenBankAF041019. FIG. 4C shows the nucleic acid sequence of human glucokinaseexon 9 with the G385V mutation (SEQ ID NO: 11). The wild-type sequenceis GenBank number AF041021.

FIG. 5 shows the nucleic acid sequence of the human amylin gene exon 3with the S20G mutation (SEQ ID NO: 12). The wild-type sequence isGenBank number X52819.

FIG. 6 shows the nucleic acid sequence base pairs 3001-3480 of the humanmitochondrion complete genome, depicting the A3243G mutation (SEQ ID NO:13). The wild-type sequence is GenBank number J01415.

FIG. 7 shows the pedigrees of families with mutations/polymorphisms inthe glucokinase (HK84) or HNF-1α gene (HK10 and HK54). Individuals withdiabetes are noted by filled symbols; individuals with impaired fastingglucose (IFG) or impaired glucose tolerance (IGT) by grey symbols;non-diabetic individuals by open symbols and untested by hatchedsymbols. The arrow indicates the proband. Present age, age at diagnosisand genotype of glucokinase or HNF-1α of tested individuals are noted:N, normal; M, mutation/polymorphism.

FIG. 8 shows the pedigrees of families with an mt3243 mutation.Individuals with diabetes are noted by filled symbols, IGT by greysymbols, non-diabetic individuals by open symbols, and untestedindividuals by hatched symbols. The arrow indicates the proband. Presentage, age of diagnosis, audiogram and genotype are also shown. N, normal;M, mutant allele.

FIG. 9A-9J show the pedigrees of 10 families carrying the HNF-1α(9A-9B), glucokinase (9C-9E), mt3243 (9F-9H) or amylin S20G (9I-9J) genemutations/polymorphisms. Subjects with diabetes are noted by blacksymbols, subjects with IFG or IGT by grey symbols, non-diabetic anduntested subjects by open symbols. The genotype of the family members isindicated by: N, wild-type allele; and M, mutant/variant allele. Presentage, age at diagnosis, therapy and complications are stated in thisorder. The proband is indicated by an arrow. Abbreviations: Oral, oraldrugs; Ins, insulin; R, retinopathy; K, albuminuria; U, neuropathy; H,hearing impairment.

FIG. 10 shows the pedigree of a Chinese family with HNF-1α IVS2nt-1G→Amutation. Subjects with diabetes are represented by black symbols,subjects with IGT by grey symbols and untested ones by open symbols. Thegenotype of family members is indicated: N, normal allele; and M, mutantallele. The proband is indicated by an arrow. CP, C-peptide; GST,glucagon stimulation test; Complications: R, retinopathy; K,nephropathy; U, neuropathy.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Compositions and methods are provided, wherein a unique combination ofgenetic markers indicative of a genetic predisposition for developingtype 2 diabetes in members of a Chinese population is described. Theinvention comprises as compositions: (1) a combination of nucleic acidsequences from wild-type genes that encode proteins important forinsulin secretory function, each nucleic acid sequence having a mutationuniquely associated with the genetic predisposition of a Chineseindividual to develop type 2 diabetes, (2) nucleic acid sequencesencoding glucokinase and HNF-1α and carrying previously unreportedmutations indicative of the increased risk of a Chinese person todevelop type 2 diabetes, (3) a microchip having attached to it at leasttwo of the mutated genes of interest, and (4) nucleic acid primers usedto detect the unique mutations in the genes of interest. The methodsinvolve: (1) obtaining genomic DNA from a Chinese subject, (2) combiningthe genomic DNA with either a combination of the mutated nucleic acidsof interest or a combination of primers used to identify the presence orabsence of a mutation in a gene of interest, and (3) detecting for thepresence or absence of mutations, either by identifying mismatchesbetween the patient's DNA and a wild-type or mutant nucleic acidsequence by hybridization techniques, or by amplifying regions of thepatients DNA that contain putative mutations by PCR, and subjecting theamplicons to restriction endonucleases and/or DNA sequencing.

Advantages of the present invention include that the method of screeninguses genetic markers shown to cosegregate with type 2 diabetes inpersons of Chinese ancestry to assess whether a given patient is atincreased risk for developing type 2 diabetes. The mutations andpolymorphisms used for screening are specifically applicable toindividuals of Chinese descent. As a further advantage, the screeningcan be based upon the presence or absence of a combination of at leasttwo different mutations or polymorphisms to provide for even moreaccurate and reliable evaluations because the contributing factors todevelopment of type 2 diabetes are heterogeneous. Identification ofparticular mutations or polymorphisms in an individual offers theadvantage that with this information physicians are able to provide morespecific and appropriate therapies for individual patients, and to guidea patient in making lifestyle adjustments to ameliorate or delaysymptoms of diabetes and associated complications. Because type 2diabetes is often an insidious disease, representative combinations ofgenetic markers indicative of a predisposition in a Chinese individualto develop the disease can be used to screen populations of individualswho may be at increased risk for developing type 2 diabetes so that theycan be given appropriate therapy before overt diabetic symptoms orcomplications are realized. Likewise, family members of an individualdiagnosed with type 2 diabetes can be screened for the particularmutants/polymorphisms of the affected individual to quickly identifyfamily members also at increased risk of developing type 2 diabetes.

By a member of a Chinese population is intended to include anyindividual of Chinese ancestry. In certain cases, for instance when amutation in a gene involved in the secretion of insulin is dominant forincreasing the risk of a Chinese individual to develop type 2 diabetes,a member of a Chinese population will encompass those individuals withat least one parent of Chinese descent. A member of a Chinese populationmay be more specifically identified by HLA haplotyping. For example, HLAclass I and class II frequencies among a Hong Kong Chinese populationhave been studied by Chang and Hawkins (Hum Immunol (1997) 56: 125).Numerous studies have been carried out to determine HLA class I andclass II alleles that are more frequently or even uniquely found inmembers of a Chinese population, and alleles with strong associations.Shaw et al and Shen et al have studied HLA polymorphism and allelefrequency and association of Chinese populations in Taiwan (TissueAntigens (1997) 50: 610; Tissue Antigens (1999) 53: 51; J Formos MedAssoc (1999) 98: 11). Allele frequency and associations found in Chineseindividuals of mainland China have been reported by Trejaut et al (Eur JImmunogenet (1996) 23: 437), Shieh, et al (Transfusion (1996) 36: 818),Zhao et al (Eur J Immunogenet (1993) 20: 293), Wang, et al (TissueAntigens (1993) 41: 223; Hum Immunol (1992) 33: 129), Lee, et al (Eur J.Immunogenet (1999) 26: 275), and Gao et al (Hum Immunol (1991) 32: 269;Tissue Antigens (1991) 38: 24; Immunogenetics (1991) 34: 401).Additionally, a Chinese individual may be objectively defined by “DNAfingerprinting” techniques well known to those in the art, wheremicrosatellite, short tandem repeat (STR) and variable number tandemrepeat (VNTR) loci specific to individuals of Chinese descent areidentified. Numerous examples of such ethnic genotyping studies havebeen reported (Meng, et al (1999) J Forensic Sci 44: 1273; Yoshimoto, etal (1999) Int J Legal Med 113: 15; Wu, et al (1999) J Forensic Sci 44:1039; Evett, et al (1996) Am J Hum Genet 58: 398; Gill and Evett (1995)Genetica 96: 69; Balazs (1993) EXS 67: 193; Lan, et al (1992) ArchKriminol 189: 169; and Hwu, et al (1992) J Formos Med Assoc 91: 839).All of these above references are incorporated herein by reference.

Whereas insulin resistance is a strong predictor of type 2 diabetes, itis not sufficient for manifestation of the disease (So, et al (2000)Hong Kong J. Med 6: 69-76). A relative insulin deficiency is essentialto the development of hyperglycemia, setting up a vicious cycle whereinelevated glucose levels are toxic to pancreatic β-cells, therebyinducing insulin resistance and decreased β-cell secretory function.Based in the intrinsic interconnection between insulin secretion andaction, the invention is exemplified by a combination of mutated genesequences from wild-type genes that are involved in insulin secretoryfunction, including hepatocyte nuclear factor 1α (HNF-1α), glucokinase,amylin and mitochondrial DNA. By “genes involved in insulin secretoryfunction” and “genes involved in insulin secretion” is intended genes inwhich a heterozygous mutation has a dominant-negative effect on normalpancreatic β-cell secretory function. The invention is primarilyconcerned with a representative array of gene markers, the combinationof which is uniquely indicative of the genetic predisposition of amember of a Chinese population to develop type 2 diabetes (referred tohereinafter as “genes of interest”). The combination of representativemutations is exemplified by G20R, A116V, IVS2nt→GA, R203H, S432C and1618M of HNF-1α; V101M, I110T, A119D, Q239R and G385V of glucokinase;S20G of amylin; and A3243G of mitochondrial tRNA^(Leu(UUR))”. Themutation IVS2nt→GA represents a splice acceptor site mutation thatlikely results in a truncated translation product.

A representative combination of the mutated genes of interest findsparticular use in the prophylactic screening of (i) Chinese individualswho have been diagnosed with maturity onset diabetes of the young (MODY)to determine the etiology of their disease, (ii) Chinese individualsthat have a positive family history of type 2 diabetes to determinetheir likelihood of developing diabetic symptoms, and (iii) and Chineseindividuals deemed to be at greater risk of developing diabetic symptomsbecause of correlative phenotypic characteristics (i. e. obeseindividuals).

The combination of the mutated genes of interest will be mostefficiently used for screening-individuals at increased risk byattaching them to a microchip or other solid support. A specific kind ofmicrochip is not critical, except that it must be able to present arepresentative array of at least two different nucleic acid sequences,each with a mutation or polymorphism indicative of the increased risk ofa Chinese individual to develop type 2 diabetes. Additionally, it willbe useful to attach representative wild-type nucleic acid sequences tothe chip as comparative controls. The microarray will normally involve aplurality of different nucleic acid sequences, usually be at least 10,more usually at least 20, frequently at least 50, but may have as manyas 100 or more. Chips that will find use with the present invention areknown in the art (for example, see U.S. Pat. Nos. 5,741,644, 5,837,832and 6,183,970). Additionally, other solid substrates may be used for thecovalent attachment of representative combinations of mutated nucleicacid sequences of interest, including beads and slides. Solid supportscan be made out of glass or silicon oxide or other materials that can beadapted to be covalently attached to oligonucleotide sequences by theintroduction of functionalities which react with oligonucleotides.

One may use a variety of approaches to bind the nucleic acid to thesolid substrate. By using chemically reactive solid substrates, one mayprovide for a chemically reactive group to be present on the nucleicacid, which will react with the chemically active solid substratesurface. For example, by using silicate esters, halides or otherreactive silicon species on the surface, the nucleic acid may bemodified to react with the silicon moiety. One may form silicon estersfor covalent bonding of the nucleic acid to the surface. Instead ofsilicon functionalities, one may use organic addition polymers. e. g.styrene, acrylates and methacrylates, vinyl ethers and esters, and thelike, where functionalities are present which can react with afunctionality present on the nucleic acid. For example, amino groups,activated halides, carboxyl groups, mercaptan groups, epoxides, and thelike, may be provided in accordance with conventional ways. The linkagesmay be amides, amidines, amines, esters, ethers, thioethers,dithioethers, and the like. Methods for forming these covalent linkagesmay be found in U.S. Pat. No. 5,565,324 and U.S. Pat. No. 6,156,501.

The invention also contemplates a microassay system and a kit thatcomprises a solid support having attached to it a representative arrayof nucleic acid sequences, each with a mutation or polymorphismassociated with the genetic disposition of a Chinese individual todevelop type 2 diabetes. The microassay system or kit would contain, forexample, a microchip or beads to which are attached wild-type and mutantnucleic acid sequences from genes that encode proteins involved ininsulin secretory function, preferentially wild-type and mutantsequences from HNF-1α, glucokinase, amylin and mitochondrial DNA.Additionally, mutant and wild-type sequences known to hybridize andknown not to hybridize under stringent conditions to those sequencesimmobilized on the support would be included as positive and negativecontrols, respectively. A microassay system or kit with nucleicsequences immobilized on a solid support would involve screening byhybridization detection (fluorescent or radioactive signal upon duplexformation). Alternatively, another microassay system or kit wouldinclude primer pairs that anneal to nucleic acid sequences encodingproteins involved in insulin secretion. The primer pairs specificallyanneal to flanking regions of the genes that putatively containmutations associated with type 2 diabetes, such that PCR amplificationwith such primers would reveal the presence or absence of an associativemutation of interes. Such a kit or microassay system would also containrepresentative mutant and wild-type sequences as controls, and screeningwould be carried out using PCR and sequencing or through PCR restrictionfragment length polymorphism analysis (RFLP) and electrophoresis.

Type 2 diabetes is a heterogeneous disease, and no single mutation orsingle mutated gene can be fully attributed to the manifestation of itssymptoms. Therefore, a combination of at least two different nucleicacid sequences encoding mutations or polymorphisms of closely associatedwith increased risk of a person of Chinese descent to develop type 2diabetes is attached to a microchip or is individually screened. By “atleast two different nucleic acid sequences” is intended two differentnucleic acid sequences from the same wild-type gene having differentmutations, or two different mutant nucleic acid sequences from twodifferent wild-type genes. Preferably, at least one of the mutant;sequences A116V of HNF-1α (SEQ ID NO: 2), V101M (SEQ ID NO: 7) or Q239R(SEQ D NO: 10) are attached to the microchip or solid support. Bywild-type gene is intended one that is not associated with type 2diabetes, and this would include any allelic variant of the wild-typegene, at any frequency, and that encodes a protein that functions in itsexpected manner without inducing pathological symptoms. By mutant geneis intended one whose sequence has been modified by insertions,deletions, or substitutions of at least one nucleic acid base pair,wherein the modification may result in detectable changes in theexpression or function of the mutant gene product as compared to thewild-type gene product. In the genes of interest for the invention, amutant gene is associated with type 2 diabetes. The nucleic acidsequences may be from genomic DNA, complementary DNA (cDNA) or frommessenger RNA (mRNA). They may be synthetic or isolated from humanbodily tissue or fluid. The mutations preferably occur, but do not needto occur, in a translated region of a nucleic acid sequence that encodesa protein that in wild-type form is involved in glucose metabolism orinsulin secretion.

Within a translated nucleic acid sequence, a mutation can be a missensemutation, replacing one amino acid with another amino acid, or anonsense mutation, replacing an amino acid with a stop codon. Mutationscan also be insertions or deletions of at least one nucleic acid ineither a coding or in non-coding region, such as a region that controlsthe transcription of a gene, including promoters, enhancers, responseelements, signal sequences and polyadenylation signals, and the like.Single nucleotide polymorphisms (SNPs), preferably but not necessarilyoccurring within the translated regions of nucleic acid sequences thatencode proteins involved in glucose metabolism or insulin secretion andthat correlate with increased risk of type 2 diabetes are alsocontemplated by the present invention. Such SNPs can be identified bycorrelating mutations in known genes that cosegregate with developmentof type 2 diabetes in members of families with a positive history of thedisease. Additionally, SNPs that occur in non-translated and translatedregions can be identified through genome-wide scans and correlatelinkage analyses of family pedigrees. The use of microarray technologiesalso can be conveniently applied to identifying SNPs of interest.

Embodiments of methods for determining or detecting the geneticpredisposition of a Chinese individual to develop type 2 diabetesinclude obtaining a sample containing genomic nucleic acid from aChinese patient, such as tissue from autopsy or biopsy, or a bloodsample, and contacting that sample with a representative combination ofat least two mutated genes of interest, then subjecting the sample DNAtogether with the patient's DNA to hybridization conditions stringentenough to detect nucleotide differences of at least one base pair.Alternatively, particular genes of interest from the genomic DNA of aChinese individual at risk are subjected to restriction fragmentationand then screened using PCR primer pairs and PCR-RFLP techniques toidentify the presence or absence of a mutation known to be associatedwith type 2 diabetes. The methods further encompass screening thegenomic DNA of Chinese individuals that have been diagnosed with type 2diabetes or that have a primary family member with type 2 diabetes foradditional associative mutations in identified genes or for mutationscorrelative with the predisposition of a member of a Chinese populationto develop type 2 diabetes in additional candidate genes, such as thoseassociated with diabetic kidney disease and obesity. Mutations are mostefficiently identified in Chinese families with a positive history ofdeveloping type 2 diabetes (i. e. families with members that developMODY). However, identified associative mutations are useful foridentifying the increased risk in any member of a Chinese population.

In practicing a method of identifying the mutations associated with thegenotype of a Chinese individual who is at increased risk for developingtype 2 diabetes, Chinese subjects with (i) a confirmed diagnosis of type2 diabetes, (ii) a positive familial history of type 2 diabetes or (iii)phenotypically determined elevated risk factors (e. g. obesity) areidentified by clinical testing, pedigree analysis, and linkage analysis,using standard diagnostic criteria and interview procedures, and DNA orRNA samples are obtained from the subjects.

A sample of genomic DNA is obtained from any nucleated cell source orbody fluid. Examples of cell sources available in clinical practiceinclude blood cells, buccal cells, cervicovaginal cells, epithelialcells from urine, fetal cells, or any cells present in tissue obtainedby biopsy. Body fluids include blood, urine, cerebrospinal fluid,amniotic fluid, and tissue exudates at the site of infection orinflammation. DNA is extracted from the cell source or body fluid usingany of the numerous methods that are standard in the art. It will beunderstood that the particular method used to extract DNA will depend onthe nature of the source.

A variety of techniques are then employed to identify the presence orabsence of new or known mutant sequences. First, the sequences of genesknown to be involved in insulin secretory function may be subjected todirect DNA sequencing, using methods that are standard in the art.Mutations may be detected using a PCR-RFLP, in which pairs ofolignucleotides are used to prime amplification reactions and the sizesof the amplification products, cleaved or uncleaved by restrictionendonucleases, are compared with those of control products. Other usefultechniques include Single-Strand Conformation Polymorphism analysis(SSCP), denaturing gradient gel electrophoresis, and two dimensional gelelectrophoresis, EMC, and the like. Detection of known mutations, suchas those exemplified by the invention, may alternatively be detectedusing nucleic acid probes that contain mutations of interest insufficiently stringent hybridization conditions.

Appropriate stringency conditions for identifying mutations of at leastone base pair in a mutant sequence of a gene involved in insulinsecretory function, for example, in 6× sodium chloride/sodium citrate(SSC) at at least 42° C., preferably at about 43,44 or 45° C., followedby a wash of 2×SSC at 50° C., are known to those skilled in the art orcan be found in Current Protocols in Molecular Biology, John Wiley &Sons, N. Y. (1989). To optimize conditions, both salt and temperaturemay be varied, or either the temperature or salt concentration may beheld constant while the other variable is changed. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.The temperature in the wash step can be increased from low stringencyconditions at room temperature, about 22° C. to high stringencyconditions at about 65° C. Optimal conditions will additionally dependon the length of the nucleic acid probe used, and the scale at which thehybridization takes place. High stringency hybridization conditionsusing nucleotides attached to a microchip may require lowertemperatures. One can perform a series of routine thermal equilibriumexperiments to determine optimal hybridization discrimination betweenwild-type and mutant gene sequences of interest, by starting a lowstringency temperature of about 20° C. and increasing the temperature insuccessive 5° C. temperature intervals.

The nucleic acid probes of the invention are nucleic acid sequences fromthe mutated genes of interest. They are at least 8,12,15 or 20 basepairs in length, but can be 50,80 or 100 base pairs in length, and mayeven be 250 or 500 base pairs in length, and include at least oneassociative mutation but may include multiple mutations, and can be aslong as the length of the transcribed gene. The length of the probechosen will be optimized based on the better base pair mismatchdiscrimination of shorter probes and the better duplex stability oflonger probes (see U.S. Pat. No. 6,156,601 and U.S. Pat. No. 6,197,506,herein incorporated by reference). The length of the probe used shouldenable discrimination between a mutant and wild-type gene with at leastone base-pair mutation.

For detection of hybridized probes, light detectable means arepreferred, although other methods of detection may be employed, such asradioactivity, atomic spectrum, and the like. For light detectablemeans, one may use fluorescence, phosphoresence, absorption,chemiluminescence, or the like. The most convenient will befluorescence, which may take many forms. One may use individualfluorescers or pairs of fluorescers, particularly where one wishes tohave a plurality of emission wavelengths with large Stokes shifts.Illustrative fluorescers which have found use include fluorescein,rhodamine, Texas red, cyanine dyes, phycoerythrins, thiazole orange andblue, etc. When using pairs of dyes, one may have one dye on onemolecule and the other dye on another molecule which binds to the firstmolecule. For example, one may have one dye on the first or boundcomponent and the other dye on the second or complexing component. Theimportant factor is that the two dyes when the two components are boundare close enough for efficient energy transfer (see U.S. Pat. No.5,992,617).

The identification of the presence or absence of known mutations canalso conveniently be detected by PCR followed by restriction analysisand/or sequencing using techniques well known to those in the art. PCRanalysis furthermore offers an efficient technique for identifying newmutations in genes already known to contain mutations that correlatewith the predisposition of a Chinese individual to develop type 2diabetes, or in identifying associative mutations in additionalcandidate genes. PCR primers should be at least 12 base pairs in length,preferably 15-18 base pairs in length, and may be as long as 25-30 basepairs in length. They can be designed to anneal to the wild-type genesequence in regions that flank a mutation in a gene of interest, suchthat extension from the primer amplifies a region that allows thedetection of the presence or absence of a mutation of interest. Primerscan also be designed such that their extension results in an amplifiedsequence only in the presence of either a wild-type or mutant gene, asdesired. This can be accomplished by designing a primer with at leastone nucleotide at the 3′end that is mismatched with the wild-typesequence, but matched to a mutant sequence. The invention is exemplifiedby primer pairs used to screen HNF-1α, glucokinase, amylin and humanmitochondrial DNA for mutations. Of particular interest are nucleic acidprimers that can be used to screen mutations in HNF-1α and glucokinasethat have not yet been previously reported (see for example, SEQ ID Nos:34-36). Simultaneous sequencing of several nucleic acid samples can alsobe carried out on a microchip (see U.S. Pat. No. 6,197,506)

For SSCP, primers are designed that amplify DNA products of about250-300 bp in length across non-duplicated segments of the gene ofinterest. For each amplification product, one gel system and two runningconditions are used. Each amplification product is applied to a 10%polyacrylamide gel containing 10% glycerol. Separate aliquots of eachamplimer are subjected to electrophoresis at 8 W at room temperature for16 hours and at 30 W at 4° C. for 5.4 hours. These conditions werepreviously shown to identify 98% of the known mutations in the CFTR gene(Ravnik-Glavac et al, (1994) Hum Mol Genet 3: 801).

As with identification of associative mutations of interest,identification of associative SNPs that correlate with the increasedrisk of a Chinese individual to develop type 2 diabetes can beaccomplished by nucleic acid sequencing of desired regions of genomic orcomplementary DNA. Screening for SNPs is pursued most efficiently usingmicroarray technologies where attached nucleic acid sequences attachedto a solid support such as a microchip are exposed to hybridizationconditions that allow the discrimination between two nucleic acidsequences that differ at one nucleotide (see for example, Wang, et al(1998) Science 280: 1077; and Hacia, et al (1998) Nature Genet. 18:155). Alternatively, mass spectrophotometers can be used to identifysmall mass differences in PCR products that have single nucleotidepolymorphisms (see Kirpekar, et al (1998) Nucleic Acids Res 26: 2554). Afurther means of analyzing genetic information is” dynamic allelespecific hybridization” (DASH). This technique uses labeledoligonucleotides in a multiwell format that will fluoresce when theoligonucleotide exists in a double-stranded form, but not when it is insingle-stranded form. Adding a single strand of the DNA to be testedallows the strands to hybridize. The temperature at which the strandsdenature will allow identification of the base at the SNP. The DASHtechnique has the advantages of being technically simple, and notrequiring expensive equipment. Additional techniques that can be used inthe screening for SNPs associated with the genetic predisposition of aChinese person to develop type 2 diabetes include exonucleaseresistance, microsequencing, solution-phase or solid phase extension ofddNTPs, and oligonucleotide ligation assay (as described in U.S. Pat.No. 5,952,174, herein incorporated by reference).

After the presence of an associative mutation or SNP is detected by anyof the above techniques, the specific nucleic acid alteration comprisingthe mutation is identified by direct DNA sequence analysis orrestriction analysis or a combination of both. In this manner,previously unidentified mutations in genes that encode proteins involvedin insulin secretion, or in genes associated with obesity or diabetickidney disease may be defined. For instance, new mutations could beidentified with other genes known to closely correlate with familialtype 2 diabetes in Chinese subjects (e.g., other MODY genes). Examplesof additional MODY genes include hepatocyte nuclear factor 4α (HNF-4α),hepatocyte nuclear factor 1β (HNF-1β), and insulin promoter factor 1(IPF-1). Additional candidate genes of particular interest for screeningbecause mutations or polymorphisms of the wild type genes are positivelyassociated with type 2 diabetes and nephropathy in Chinese individualsinclude those that encode angiotensin converting enzyme(ACE)/angiotensinogen (AGT) (Tomino, et al (1999) Nephron 82: 139;Hsieh, et al (2000) Nephrol Dial Transplant 15: 1008; Thomas, et al(2001) Diabetes Care 24; 356), aldose reductase (Ko, et al (1995)Diabetes 44: 727; Moczulski, et al (1999) Diabetologia 42: 94) andplasminogen activator inhibitor-1 (PAI-1) (Wong, et al (2000) Kidney Int57: 632).

Nucleic acid sequences that encode genes involved with glucosemetabolism, insulin resistance, obesity and diabetic kidney can also bescreened to identify mutations in, for example, proteins that influenceinsulin binding to its receptor, that are involved in the insulinsignaling pathway, that influence glucose uptake and cell metabolism.Specific examples include associative mutations in the α or β chain ofthe insulin receptor, the insulin receptor substrate proteins (IRS-1 andIRS-2), glucose transporter proteins GLUT2 and GLUT4, and transcriptionfactors HNF-3β and NeuroDI/Beta2 and to correlated any identifiedmutation an/or polymorphism with indidence of type 2 diabetes. Examplesof candidate genes where mutations or polymorphisms have been shown tobe associated with type 2 diabetes and obesity in other populationsinclude genes that encode the transporter GLUT4 (Abel, et al (2001)Nature 409: 729), the beta-3-adrenergic receptor (Oeveren van-Dybicz, etal (2001) Diabetes Obes Metab 3: 47), the hormone resistin (Steppan, etal2001) Nature 409: 307), the peroxisome proliferator-activated receptorgamma2 PPARgamma) (Hasstedt, et al (2001) J Clin Endocrinol Metab 86:536), uncoupling protein-1 (UCP-1) (Heilbronn, et al (2000) Diabetologia43: 242), leptin (Ohshiro, et al (2000) J Mol Med 78: 516), G proteinbeta 3 subunit and insulin receptor substrate-1 (Rosskopf, et al (2000)5: 484), and the dopamine D2 receptor (Jenkinson, et al (2000) Int JObes Relat Metab Disord 24: 1233). Additionally, mutations orpolymorphisms shown to be closely associated with type 2 diabetes andnephropathy in other populations include genes that encode the G proteinbeta 3 subunit (Beige, et al (2000) Nephrol Dial Transplant 15: 1384;Zychma, et al (2000) Am J Nephrol 20: 305), methylenetetrahydrofolatereductase (MTHFR) (Shpichinetsky, et al (2000) J Nutr 130: 2493, theglucose transporter GLUT1 (Grzeszczak, et al (2001) Kidney Int 59: 631),and paraoxonase (PON1) (Inoue, et al (2000) Metabolism 49: 1400).

Additionally, proteins translated from genes carrying at least onemutation associated with increased risk of a Chinese individual todevelop type 2 diabetes are contemplated by the invention and find usein functional diagnostic assays and in the production of diagnosticantibodies that bind to the mutant but not the wild-type protein. Thepolypeptides may be the translational products of the entire mutantgene, as well as peptides of twelve or more amino acids derivedtherefrom that contain at least one mutation of interest. Thepolypeptide (s) may be isolated from human tissues obtained by biopsy orautopsy, or may be produced in a heterologous cell by recombinant DNAmethods, well known to those in the art (as disclosed in MolecularCloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis,Cold Spring Harbor, 1989), or Current Protocols in Molecular Biology(Eds. Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl,GreenePubl. Assoc., Wiley-Interscience, NY, N.Y., 1992), both referencesherein incorporated by reference). Peptides comprising HNF-1α-,glucokinase- or amylin specific sequences may be derived from isolatedlarger polypeptides described above, using proteolytic cleavages by e.g. proteases such as trypsin and chemical treatments such as cyanogenbromide that are well-known in the art. Alternatively, peptides up to 60residues in length can be routinely synthesized in milligram quantitiesusing commercially available peptide synthesizers.

Recombinant translational products are expressed from vectors comprisingmutant nucleic acid sequences of wild-type nucleic acid sequences thatencode proteins involved in insulin secretion. Exemplified mutantnucleic acid sequences of interest include those that encode HNF-1α,glucokinase or amylin with single amino acid residue changes, asdepicted in SEQ ID Nos: 1-13, and particularly SEQ ID NO: 2, SEQ ID NO:7 and SEQ ID NO: 10. A large number of vectors, including plasmid andfungal vectors, have been described for expression in a variety ofeukaryotic and prokaryotic hosts, and may be used for gene therapy aswell as for simple protein expression. Vectors used for expression willalso include a promoter operably linked to the mutant polypeptideencoding portion, that is preferably the cDNA sequence of the mutatedgene of interest or a part thereof that encodes a polypeptide of atleast 12 amino acids. The encoded polypeptide may be expressed by usingany suitable commercially available vectors, and any suitable hostcells, using methods disclosed or cited herein or otherwise known tothose skilled in the relevant art. The particular choice of vector/hostis not critical to the operation of the invention.

Appropriate host cells include bacteria, archebacteria, fungi,especially yeast, and plant and animal cells, especially mammaliancells. Of particular interest are E.coli, B.Subtilis, Saccharomycescerevisiae, SF9 cells, C129 cells, 293 cells, Neurospora, and CHO cells,COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoidcell lines. Preferred replication systems include M13, ColEI, SV40,baculovirus, lambda, adenovirus, and the like. A large number oftranscription initiation and termination regulatory regions have beenisolated and shown to be effective in the transcription and translationof heterologous proteins in the various hosts. Examples of theseregions, methods of isolation, manner of manipulation, etc. are known inthe art. Under appropriate expression conditions, host cells can be usedas a source of recombinantly produced mutant polypeptides of interest.

The translational products of mutant HNF-1α, glucokinase or amylin,and/or fragments or portions thereof may be used to produce specificantibodies. The antibodies may be polyclonal or monoclonal, may beproduced in response to the fully translated mutant polypeptide or tosynthetic peptides as described above. Such antibodies are convenientlymade using the methods and compositions disclosed in Harlow and Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988,other references cited herein, as well as immunological and hybridomatechnologies known to those in the art. Importantly, the antibodiesraised against translation products from nucleic acids sequencescarrying at least one mutatation associated with type 2 diabetes shoulddistinguish between the mutant amino acid sequence and the wild-typeamino acid sequence. In particular, antibodies should have very littleor no cross-reactivity for the wild-type sequence. Preferably theanti-mutant protein antibodies should bind with higher affinity to themutant polypeptide than to wild-type polypeptide, with binding to themutant polypeptide at levels 500:1, more preferably 1,000:1, greaterthan binding the wild-type polypeptide.

Isolated polypeptides corresponding to the entire length of the mutantpolypeptide or a peptide of at least 12 amino acids in length containinga mutation of interest may be used in accordance with conventionalmethods to immunize a mammal, (e. g., mouse or higher mammal, primate,or chimeric or transgenic animals which produce human immunoglobulins)in accordance with conventional procedures. See for example, U.S. Pat.Nos. 4,172,124; 4,350,683; 4,361,549; and 4,464,465. Hybridomas may beprepared by fusing available myeloma lines, e. g., NS/I, Ag8.6.5.3,etc., with peripheral blood lymphocytes, splenocytes or otherlymphocytes of the immunized host and the resulting immortalizedB-lymphocytes (e.g., hybridomas, heteromyelomas, EBV transformed cells,etc.) selected, cloned and screened for binding to a mutant polypeptideof a wild-type protein involved in insulin secretion or glucosemetabolism. Monoclonal antibodies raised against a mutant polypeptidesequence of interest may be of any immunoglobulin class such as IgA,IgD, IgE, IgG and IgM, preferably IgG or IgM, and may be of any one ofthe subclasses of the classes. Whole antibodies, or fragments thereofwhich retain binding activity, may be employed, such as Fab, F(ab′)₂, orthe like. Once the antibodies with binding specificity for the mutantpolypeptide are available, these antibodies may be used for screening.Antibodies that distinguish between normal and mutant forms of HNF-1α,glucokinase, amylin or other mutant/wild-type pairs of proteins involvedin insulin secretory function may be used in diagnostic tests employingELISA, EMIT, CEDIA, SLIFA, and the like.

For an assessment of total risk of developing disease or in designingindividualized treatments of diagnosed patients, identified mutationsand polymorphisms that are indicative of a Chinese individual to developtype 2 diabetes are correlated with phenotypic parameters of screenedpatients and interpreted with consideration of a positive or negativefamily history of the disease. Genetic studies will be correlated withdata from individuals indicating hormone levels (growth hormone,adrenaline, cortisol, noradrenaline, insulin), anthropometry (body-massindex; waist-to-hip ratio), hemodynamics (blood pressure),cardiovascular risk factors (HDL, LDL, cholesterol, triglycerides) andautoimmunity (anti-glutamic acid decarboxylase antibodies). Forinstance, a patient with a single mutation in the glucokinase gene maynever develop symptoms, whereas the likelihood of a patient with amutation in both the glucokinase gene and the HNF-1α gene or a mutationin the glucokinase gene and the phenotypic attribute of obesity todevelop overt type 2 diabetes is relatively higher. A positive familyhistory of the disease would increase the predicted predisposition evenmore. Obtaining a genotypic assessment while a patient shows no signs ofdeveloping disease, or while showing preliminary signs of disease suchas impaired glucose tolerance (IGT), can enable a physician to initiatetherapy or suggest lifestyle changes that prevent the onset orprogression of overt symptoms. For example, a patient identified ashaving a mutation in the HNF-1α gene and IGT, can be treated with dietand/or oral drugs and/or insulin early enough that hyperglycemictoxicity of pancreatic β-cells and further insulin secretory dysfunctiondue to their death is prevented or ameliorated. In this way, severecomplications associated with progressive type 2 diabetes, such asnephropathy, retinopathy and sensorineural loss, can be more commonlyaverted.

In addition to allowing a clinician to better tailor traditionaltherapies for treating type 2 diabetes, such as diet, oral drugs andinsulin, identification of associative mutations can enable a clinicianto design tailored therapies, such as introducing a wild-type gene intoa patient to replace a mutant gene that encodes a malfunctioningprotein. For gene therapy methods, transfection in vivo is obtained byintroducing a therapeutic transcription or expression vector into themammalian host, either as naked DNA, complexed to lipid carriers,particularly cationic lipid carriers, or inserted into a viral vector,for example a recombinant adenovirus. The introduction into themammalian host can be by any of several routes, including intravenous orintraperitoneal injection, intratracheally, intrathecally, parenterally,intraarticularly, intranasally, intramuscularly, topically,transdermally, application to any mucous membrane surface, cornealinstallation, etc. Of particular interest is the introduction of atherapeutic expression vector into a circulating bodily fluid or into abody orifice or cavity, such as the heart. Thus, intravenousadministration and intrathecal administration are of particular interestsince the vector may be widely disseminated following such routes ofadministration, and aerosol administration finds use with introductioninto a body orifice or cavity. Particular cells and tissues can betargeted, depending upon the route of administration and the site ofadministration. For example, a tissue which is closest to the site ofinjection in the direction of blood flow can be transfected in theabsence of any specific targeting. An arterial catheter can be used tointroduce the expression vector into an organ such as the heart orkidney. The eye can be accessed directly either by the use of oculardrops or by injecting into the eye. For accessing nerves, this can be byinjection into the nerve or injection into the region of the cell body.If lipid carriers are used, they can be modified to direct the complexesto particular types of cells using site-directing molecules. Thus,antibodies or ligands for particular receptors or other cell surfaceproteins may be employed, with a target cell associated with aparticular surface protein. An amino terminal mitochondrial targetingsequence joined to a nucleic acid can be used to target the nucleic acidto the mitochondria. See Taylor et al, Nature Genetics 15: 212-215,1997.

Any physiologically acceptable medium may be employed for administeringthe DNA, recombinant viral vectors or lipid carriers, such as deionizedwater, saline, phosphate-buffered saline, 5% dextrose in water, and thelike as described above for the pharmaceutical composition, dependingupon the route of administration. Other components may be included inthe formulation such as buffers, stabilizers, biocides, etc. Thesecomponents have found extensive exemplification in the literature andneed not be described in particular here. Any diluent or components ofdiluents that would cause aggregation of the complexes should beavoided, including high salt, chelating agents, and the like.

The amount of therapeutic vector used will be an amount sufficient toprovide for a therapeutic level of expression in a target tissuesusceptible to diabetic complications or for adequate dissemination to avariety of tissues after entry into the bloodstream and to provide for atherapeutic level of expression in susceptible target tissues. Atherapeutic level of expression is a sufficient amount of expression toprevent, treat, or palliate one or more diabetic complication or thesymptoms of diabetic complications. In addition, the dose of the nucleicacid vector used must be sufficient to produce a desired level oftransgene expression in the affected tissue or tissues in vivo. OtherDNA sequences, such as adenovirus VA genes can be included in theadministration medium and be co-transfected with the gene of interest.The presence of genes coding for the adenovirus VA gene product maysignificantly enhance the translation of mRNA transcribed from theexpression cassette if this is desired.

A number of factors can affect the amount of expression in transfectedtissue and thus can be used to modify the level of expression to fit aparticular purpose. Where a high level of expression is desired, allfactors can be optimized, where less expression is desired, one or moreparameters can be altered so that the desired level of expression isattained. For example, if high expression would exceed the therapeuticwindow, then less than optimum conditions can be used.

The level and tissues of expression of the recombinant gene may bedetermined at the mRNA level as described above, and/or at the level ofpolypeptide or protein. Gene product may be quantitated by measuring itsbiological activity in tissues. For example, protein activity can bemeasured by immunoassay as described above, by biological assay such asinhibition of ROS, or by identifying the gene product in transfectedcells by immunostaining techniques such as probing with an antibodywhich specifically recognizes the gene product or a reporter geneproduct present in the expression cassette.

Typically, the therapeutic cassette is not integrated into the patient'sgenome. If necessary, the treatment can be repeated on an ad hoc basisdepending upon the results achieved. If the treatment is repeated, thepatient can be monitored to ensure that there is no adverse immune orother response to the treatment.

The following examples are offered by way of illustration of the presentinvention, not limitation.

EXPERIMENTAL EXAMPLE 1 Identification of Mutations in Glucokinase andHepatocyte Nuclear Factor 1α Genes in Chinese Patients with Early-OnsetType 2 Diabetes Mellitus/MODY

This example illustrates mutations identified in the glucokinase, HNF-1αand HNF-4α genes in a cohort of Chinese patients. Mutations in theglucokinase and HNF-1α genes are relatively common in early-onsetdiabetes and they account for about 3% and 5%, respectively, of thepresent Chinese early-onset diabetic patients.

Experimental Design and Methods

Subjects

The study group consisted of 92 unrelated patients (age 34±5 years(mean±SD) range 18-40 years; 30 males and 62 females) who were diagnosedwith Type 2 diabetes before 40 years of age and who had a positivefamily history (at least one first degree relative with Type 2diabetes). The mean age at diagnosis was 30±5 years (range 16-40 years).Thirteen (14%) of these patients met the minimal criteria of MODY (ageat diagnosis before 25 years old and presence of diabetes in twoconsecutive generations). These patients were selected from a databasecontaining 1800 cases recruited in the Diabetes and Endocrine Centre ofthe Prince of Wales Hospital. Family members of probands with MODY genemutations, if available, were recruited and underwent a 75-gram oralglucose tolerance test (OGTT). One hundred healthy Chinese (age 33±10years, 40 males and 60 females) without a history of diabetes wererecruited as controls amongst hospital staff and students. Informedconsent was obtained from each subject for a blood sample to be takenfor DNA isolation and measurement of clinical parameters. This study wasapproved by the Clinical Research Ethics Committee of The ChineseUniversity of Hong Kong.

Screening of Glucoknase, HNF-1α and HNF-4α Genes for Mutations

The minimal promoter region and exons of the glucokinase (β cell form),HNF-1α and HNF-4α (HNF-4α 2 form) genes were screened for mutations bydirect sequencing of polymerase chain reaction (PCR) products asdescribed (Froguel, et al (1993) N Engl J Med 328: 697; Yamagata, et al(1996) Nature 384: 455; Yamagata, et al (1996) Nature 384: 458). Theoccurrence of putative mutations in other family members and controlswas determined by PCR-restriction fragment length polymorphism (RFLP).An artificial restriction site was introduced into either the wild-typeor mutant sequence if the nucleotide substitution did not lead to gainor loss of a restriction site. Briefly, direct sequencing identified 5mutations in the HNF-1α gene (G20R, R203H; S432C, I618M and IVS2nt-1G→A)and 3 mutations in the glucokinase gene (I110T, A119D and G385V) thatare unique to Chinese subjects. They were screened as follows.

HNF-1α G20R was screened by using the forward primer5′-GGCAGGC-AAACGCAACCCACG-3′ (SEQ ID NO:14) and modified reverse primer5′-CAGTGCCTCTTTGCTCAGGC-3′ (SEQ ID NO:15) for PCR amplification followedby digestion with StuI. The wild-type allele showed 19 and 140 bpproducts whereas the mutant allele showed a 159 bp product.

HNF-1α R203H was screened by using the forward primer5′-TGCCTGCAGAGTTCA-CCCATG-3′ (SEQ ID NO:16) and modified reverseprimer5′-ATCTGCTGGGATGCTGGG-CCCCACTTGCAA-3′ (SEQ ID NO:17) for PCRamplification followed by digestion with BsrDI. The wild-type alleleshowed a 121 bp product whereas the mutant allele showed 26 and 95 bpproducts.

HNF-1α S432C was screened by using the forward primer5′-TGGAGCAGTCCCTAG-GGAGGC-3′ (SEQ ID NO:18) and reverse primer5′-GTTGCCCCATGAGCCTCCCAC-3′ (SEQ ID NO:19) for PCR amplificationfollowed by digestion with Cac81. The wild-type allele showed 104 and218 bp products whereas the mutant allele showed 37,67 and 218 bpproducts.

HNF-1α 1618M was screened by using the forward primer5′-GTACCCCTAGGGACAGG-CAGG-3′ (SEQ ID NO:20) and reverse primer5′-ACCCCCCAAGCAGGCAGTACA-3′ (SEQ ID NO:21) for PCR amplificationfollowed by digestion with TaqI. The wild-type allele showed 88 and 160bp products whereas the mutant allele showed a 248 bp product.

HNF-1α IVS2nt-1G→A was screened by using the forward primer5′-GGGC-AAGGTCAGGGGAATGGA-3′ (SEQ ID NO:22) and reverse primer5′-CAGCCCAGACCAAACCAGCAC-3′ (SEQ ID NO:23) for PCR amplificationfollowed by digestion with PstI. The wild-type allele showed 73 and 231bp products whereas the mutant allele showed a 304 bp product.

Glucokinase I110T was screened by using the forward primer5′-GTCCCTGAGGCTG-ACACACTT-3′ (SEQ ID NO:24) and reverse primer5′-AGCTGGGCCCTGAGATCCTGCA-3′ (SEQ ID NO: 25) for PCR amplificationfollowed by digestion with FokI. The wild-type allele showed 108 and 142bp products whereas the mutant allele showed a 250 bp product.

Glucokinase A 119D was screened by using the forward primer5′-ACCTGGGTGGCA-CTAACTTCA-3′ (SEQ ID NO:26) and modified reverse primer5′-CGGCCCCTGCGCTG-CTCACCATCTGA-3′ (SEQ ID NO:27) for PCR amplificationfollowed by digestion with BclI. The wild-type allele showed a 150 bpproduct whereas the mutant allele showed 28 and 122 bp products.

Glucokinase G385V was screened by using the forward primer5′-GGACTGTCG-GAGCGACACTCA-3′ (SEQ ID NO:28) and modified reverse primer5′-GCGGTTGATGAC-GCCTGCCAG-3′ (SEQ ID NO:29) for PCR amplificationfollowed by digestion with FauI. The wild-type allele showed 5,22,44 and137 bp products whereas the mutant allele showed 5,44 and 159 bpproducts.

Mutations in the amylin gene (S20G) and mitochondrial DNA (A3243G) werescreened as follows.

Amylin S20G was screened by using the forward primer5′-TCACAT-TTGTTCCATGTTAC-3′ (SEQ ID NO:30) and reverse primer5′-CAATAACTATAGAG-TTACATTG-3′ (SEQ ID NO:31) for PCR amplificationfollowed by digestion with MspI. The wild-type allele showed a 239 bpproduct whereas the mutant allele showed 99 and 140 bp products.

Mitochondrial DNA A3243G was screened by using the forward primer5′-AAGGTTCGTT-TGTTCAACGA-3′ (SEQ ID NO:32) and reverse primer5′-AGCGAAGGGTTGTAGTAGCC-3′ (SEQ ID NO:33) for PCR amplification andlabeling of PCR product with α³²PdATP at the last cycle. The PCRproducts were then digested with ApaI and analysed on 8% denaturingpolyacrylamide gels. The wild-type allele showed a 427 bp productwhereas the mutant allele showed 213 and 214 bp products.

Clinical Studies

All patients underwent a structured assessment including documentationof family history, age at diagnosis and body mass index (BMI)(Piwernetz, et al (1993) Diabetic Med 10: 371; Chan, et al (1997) HongKong Auth Qual Bull 2: 3). Family history was documented in twogenerations only since the diabetic status of grandparents was usuallyunknown. A fasting blood sample was taken for the measurement ofglucose, C-peptide and glycosylated haemoglobin (HbA_(1c)). Obesity wasdefined as a BMI≧27 kg/m²in men and ≧25 kg/m²in women (National DiabetesData Group (1979) Diabetes 28: 1039).

Assays

Plasma glucose concentrations were measured by a glucose oxidase method(Diagnostic Chemicals, Charlottetown, Prince Edward Island, Canada).C-peptide was measured by radioimmunoassay (Novo-Nordisk, Copenhagen,Denmark). HbA_(1c) was measured by gel electrophoresis (Ciba ComingDiagnostics Corp, Palo Alto, Calif.).

Data Analysis

Data are expressed as mean ±SD if normally distributed. Otherwise, dataare expressed as median and range.

Results

Mututions and Polymorphisms in the Glucokinase, HNF-1_(α) and HNF-4_(α)Genes

Screening of the promoter region and exons 1a, 2-10 of-the glucokinasegene (Stoffel, et al (1992) Proc Nratl Acad Sci 89: 7698; Tanizawa, etal (1992) 6:1070) revealed three novel missense mutations: I110T, A119Dand G385V. In addition to these mutations, three uncommon variants (twoof which had not been previously described) and two polymorphisms werefound in the 5′-untranslared region of the mRNA and intron regions(Table 1). The brother and mother of subject HK84 (Table 1) alsoinherited the I110T mutation (FIG. 7). The mother was diagnosed withdiabetes at the age of 64 years upon screening. The brother aged 25years, when tested with a 75 g OGTT had a plasma glucose at 0 and 120min of 6.3 mmol/1 and 6.9 mmol/1, respectively. These results wereinconclusive, suggesting impaired fasting glucose (IFG) by the 1997 ADAcriteria but not reaching that of impaired glucose tolerance (IGT) bythe 1998 WHO criteria.

Screening of the HNF-1α gene revealed four missense mutations (G20R,R203H, S432C and 1618M) and one splice acceptor site mutation(IVS2nt-1G→A) (Table 2). All of these represent mutations in the HNF-1αgene unique to Chinese patients. Subject HK10 (Table 2) had threesiblings (ages 26-36 years) with diabetes. The affected siblings all hadinherited the IVS2nt-IG→A mutation while another sibling and the fatherwith IGT had not. Moreover, the maternal grandparents, uncle and motherof HK10 were diabetic but they were not available for screening (FIG. 7)(Chan, et al (1990) Diabetic Med 7: 211). Subject HK54 (Table 2) hadfour siblings (age 33-43 years) with normal glucose tolerance and onesibling (age 39 years) with IGT. The father and mother were diagnosed ashaving diabetes at the ages of 50 and 60 years, respectively. Neitherthe mother and nor any of the siblings had inherited the R203H mutation(FIG. 7). In addition to the putative diabetes-associated mutations inHNF-1α two substitutions resulting in common amino acid polymorphisms,four silent mutations and nine variants/polymorphisms in introns wereidentified (Table 2). Family members of the other five probands (Tables1 and 2) with glucokinase or HNF-1α missense mutations were notavailable for screening. None of the mutations in the glucokinase andHNF-1α genes were found in 100 healthy controls.

Analysis of the promoter region and exons 1a, 2-10 of the HNF-4α gene(Furuta, et al (1997) Diabetes 46: 1652) revealed no obviousdiabetes-associated mutations. Three patients were heterozygous for apreviously described amino acid polymorphism, T/1130 (Yamagata et al,(1997) Nature 384: 458). Two patients were heterozygous for a silentmutation in the codon for L211, and one patient was heterozygous for asilent mutation in the codon for P441 (Table 3). There were twopolymorphisms in the intron-upstream of exon 2 (intron 1B) and a G→Asubstitution in the promoter was found in the heterozygous state in onepatient. The G→A substitution in the promoter at nucleotide-462 was notlocated in a known cis-acting regulatory region of the gene (Furuta, etal (1997) supra) and its effect on the regulation of expression ofHNF-4α remains to be determined.

Clinical Features of Patients with MODY or Unknown Etiology

The clinical features of the patients with mutations in the glucokinaseand HNF-1α genes or with unknown etiology are shown in Table 4. Of the92 patients, 54 (59%) were non-obese at the time of study. The mean ageat diagnosis of the patients with glucokinase mutation-associateddiabetes (‘glucokinase diabetes’) was 28 years. All three subjects hadmild hyperglycemia and satisfactory glycemic control (fasting glucose≦7.4 mmol/1; HbA_(1c), 6.7%; non-diabetic range: 5.1-6.4%). Thesepatients had varving degrees of basal pancreatic β cell secretoryfunction as indicated by their fasting C-peptide levels (0.28-1.60nmol/1) (Chan, et al (1990) supra). All were treated with diet or oraldrugs. No diabetic complications were observed in the three patientswith glucokinase mutation (Froguel, et al (1993) supra ; Page, et al(1995) 12: 209; Velho, et al (1997) 40: 217).

The mean age at diagnosis of the patients with HNF-1αmutation-associated diabetes was 31 years. Among the four patients(HK30, 54,90 and 92) with missense mutations, all had mild hyperglycemiaand satisfactory glycemic control (fasting glucose ≦7.4 mmol/1;HbA_(1c)≦7.1%) but exhibited varying degrees of basal pancreatic β cellsecretory function (fasting C-peptide, 0.10-0.49 nmol/1). They did nothave diabetic complications and were treated with diet or oral drugs.The subject (HK10) with the splice-site IVS2nt-1G→A mutation was notoverweight when diagnosed at the age of 19 years (Fajans (1990) DiabetesCare 13: 49-64) and presented with proliferative retinopathy andclinical proteinuria. She was treated with insulin continuously forthree months after the diagnosis. She eventually developed neuropathyand renal failure. TABLE 1 Mutations and polymorphisms in theglucokinase gene in Chinese subjects with early-onset Type 2 diabetesmellitus Subject Location Codon/nt Nucleotide change DesignationFrequency Mutations HK84 Exon 3  110 ATC (Ile)→ACC (Thr) I110T HK38 Exon3  119 GCT(Ala)→GAT(Asp) A1I9D HK15 Exon 9  385 GGG(Gly)→GTG(Val) G385VPolymorphisms 5′-UT* −213 A→G 5′-UT β − 213 A/G A 0.96, G 0.04 5′-UT −84 C→G 5′-UT β − 84 C/G C 0.94, G 0.06 Intron 1c nt − 13 C→G IVS1nt −13 C/G C 0.99, C 0.01 Intron 9 nt + 8 C→T IVS9nt + 8C/T C 0.50, T 0.50Intron 9* nt + 49 G→A IVS9nt + 49G/A G 0.99, A 0.01nt indicates the nucleotide location relative to the first nucleotide ofcodon 1 (ATG) for polymorphisms in the 5′-untranslated region (5′-UT) ofthe β cell specific exon 1 α/1 β, and splice donor (+) or acceptor site(−).Intron 1c is the intron between exon 1c, which encodes the aminoterminal 14 amino acids of the minor liver isoform of glucokinase, andexon 2 (Velho, et al (1996) 19: 915).The asterisks indicate polymorphisms that were reported by Ng, et al(Diabetic Med (1999) 16: 956) and that have not been reported in studiesof other populations (Veiga-deCunha, et al (1996) J Biol Chem 271: 6292;Zhang, et al (1995) 38: 1055).

TABLE 2 Mutations and polymorphisms in the HNF-1 α gene in Chinesesubjects with early-onset Type 2 diabetes mellitus Subject LocationCodon/n Nucleotide change Designation Frequency Mutations HK90 Exon 1 20 GGG (Gly)→AGG (Arg) G20R HK10 Intron 2/ nt − 1 AG→AA at spliceIVS2nt − 1G→A Exon 3 acceptor site HK54 Exon 3 203 CGT (Arg)→CAT (His)R203H HK30 Exon 6 432 TCC (Ser)→TGC (Cys) S432C HK92 Exon 10 618 ATC(Ile)→ATG (Met) I618M Silent mutations/polymorphisms Exon 1  17 CTC(Leu)→CTG (Leu) L17C/G C 0.63, C 0.37 Exon 1  27 ATC (Ile)→CTC (Leu)I/L27 A 0.57, C 0.43 Intron 1 nt − 42 G→A IVS1nt − 42G/A C 0.58, A 0.42Intron 2* nt + 53 C→G IVS2nt + 53C/G C 0.99, C 0.01 Intron 2 nt − 51 T→AIVS2nt − 51T/A T 0.77, A 0.23 Intron 2 nt − 23 C→T IVS2nt − 23C/T C0.48, T 0.52 Intron 5 nt + 9 C→G IVS5nt + 9C/G C 0.98, G 0.02 Intron 5nt − 42 G→T IVS5nt − 42G/T G 0.87, T 0.13 Intron 6* nt + 26 C→T IVS6nt +26C/T C 0.99, T 0.01 Exon 7 459 CTG (Leu)→TTG(Leu) L459C/T C 0.48, T0.52 Exon 7 459 CTG (Leu)→CTA (Leu) L459G/A C 0.99, A 0.01 Exon 7 487AGC (Ser)→AAC (Asn) S/N487 G 0.48, A 0.52 Intron 7 nt + 7 G→A IVS7nt +7G/A G 0.48, A 0.52 Exon 8* 531 AGC (Ser)→AGT (Ser) S531 C/T C 0.99, T0.01 Intron 9 nt − 24 T→C IVS9nt − 24T/C T 0.48, C 0.52nt indicates the nucleotide location relative to the splice donor (+) oracceptor site (−).The asterisks indicate polymorphisms reported by Ng, et al (Diabetic Med(1999) 16: 956) and that have not been reported in studies of otherpopulations.

TABLE 3 Mutations and polymorphisms in the HNF-4 α gene in Chinesesubjects with early-onset Type 2 diabetes mellitus Location Codon/ntNucleotide change Designation Frequency Silent mutations/ polymorphismsPromoter* nt-462 G→A Ptr-462G/A G 099, A 0.01 Intron lB nt-38 C→TIVS1nt-38C/T C 0.80, T 0.20 Intron lB nt-5 C→T IVS1nt-5C/T C 0.79, T0.21 Exon 4 130 ACT (Thr)→ATT (Ile) T/I130 C 0.98, T 0.02 Exon 6* 211CTC (Leu)→CTT (Leu) L211C/T C 0.99, T 0.01 Exon. 10* 441 CCG (Pro)→CCA(Pro) P441G/A G 0.99, A 0.01 G 0.99, A 0.01nt indicates the nucleotide location relative to the first nucleoride ofcodon 1 (ATG) for polymorphisms in the promoter region and splice donor(+) or acceptor site (−).The sequence context of the nt-464 polymorphism is GATA(G/A)TATC.The asterisks indicate polymorphisms that have not been reported instudies of other populations.

TABLE 4 Clinical features of Chinese patients with early-onset Type 2diabetes of unknown etiology as compared with those with diabetes as aresult of mutations in glucokinase and HNF-1 α genes Unknown GlucokinaseEtiology diabetes (n = 3) HNF-1 α diabetes (n = 5) (n = 84) HK1 HK38HK84 HK10 HK30 HK54 HK90 HK92 Age at diagnosis (year) 30 ± 5  18   29 3819 30 33 36 38 Interval since diagnosis (year) 3 (0-16) 15   2 1 9 3 6 00 Sex(M/F)(%) 32/68 F F F F M F M M Family history(fa/mot/sib)(%)45/63/25 fa mot, sib mot gparent, fa fa, mot mot, sib mot uncle, mot,sib BMI (kg/m²) 26 ± 5   nd* 15 28 26 23 20 20 29 HbA_(1c)(%) 7.5 ± 1.96.7 6.0 6.6 8.7 7.1 6.0 6.9 6.1 Fasting glucose (mmol/l) 8.5 ± 3.3 7.27.4 6.6 13.9 7.4 4.9 4.9 6.6 Fasting C-peptide (nmol/l)  0.43(0.03-4.96) nd 1.60 0.28 0.47 0.49 0.11 0.16 0.10 Treatment (D/O/I)(%)49/43/8  O O D I O O D Ogparent, grandparent affected;uncle, uncle affected;fa, father affected;mot, mother affected;sib, siblings affected;D, diet;O, oral drugs;I, insulin;nd, not done.Data are expressed as mean ± SD median (range) or n.*BMI was not measured because this patient had spinomuscular atrophy.

The mean age at diagnosis of the 84 patients with unknown etiology was30 years, similar to those with glucokinase or HNF-α gene mutations.Large variations in the degree of hyperglycemia (fasting glucose 8.5±3.3mmol/1) and basal pancreatic β cell secretory function (fastingC-peptide 0.03-4.96 nmol/1) were observed. Most of these pateints weretreated with oral drugs or diet (92%).

EXAMPLE 2 Mitochondrial DNA A3243G Mutation in Patients with Early-orLate-Onset Type 2 Diabetes Mellitus in Hong Kong Chinese

This example illustrates the prevalence of the mitochondrial DNA A3243Gmutation in the Hong Kong Chinese population as represented by a largecohort of type 2 diabetic patients with differing ages of diagnosis andclinical phenotypes.

Experimental Design and Methods

Subjects

The study group consisted of 906 unrelated type 2 diabetic patientsdiagnosed according to the 1985 WHO criteria (World Health Organization,1985). This cohort included four groups of patients selected accordingto the age of diagnosis and the presence or absence of family history ofdiabetes. Groups 1 and 2 consisted of 219 and 128 patients,respectively, with an early age of diagnosis (≦40 years) and with(Group 1) or without (Group 2) a family history of diabetes. Groups 3and 4 consisted of 211 and 348 patients, respectively, with an older ageof diagnosis (>40 years) and with (Group 3) or without (Group 4) afamily history of diabetes. Patients in each of these groups wererandomly selected from a cohort recruited in the Diabetes and EndocrineCentre of the Prince of Wales Hospital, which has a catchment of 1.2million population in Hong Kong. All the patients underwent a structuredassessment based on the Europe DiabCare Protocol (Piwernetz, et al(1993) Diabetic Med 10: 371; Chan, et al (1997) Hosp Auth Qual Bull 2:3). Family members of mt3243 mutation carriers, if available, wererecruited and underwent a 75 grain oral glucose tolerance test (OTT) Twohundred and thirteen healthy Chinese without a history of diabetes wererecruited as control subjects amongst hospital staff and students. Thepresent study group included 75 early onset type 2 diabetic (two of whomhad an mt3243 mutation) and 95 control subjects who were included in aprevious report (see Smith, et al (1997) Diabetic Med 14: 1026).Informed consent was obtained from each subject for a blood sample to betaken for DNA isolation and measurement of clinical partners. This studywas approved by the Clinical Research Ethics Committee of The ChineseUniversity of Hong Kong.

Mt3243 Mutation Analysis

Leukocyte DNA was extracted by standard methods involving proteinase Kand phenol/chloroform (Sambrook, et al (1989) Molecular Cloning: ALaboratory Manual,Cold Spring Harbor Laboratory Press, New York,incorporated herein by reference). The mt3243 genotype was determined bypolymerase chain reaction (PCR) amplification and ApaI digestion asdescribed (Smith, et al, (1997) supra). In brief, the DNA regionspanning nucleotide 3029 and 3456 was amplified by PCR and labelled withα³²PDATP at the last cycle. This method prevents the underestimation ofthe proportion of mutant mtDNA as a consequence of heteroduplexformation during the PCR (Schoffner, et al (1990) Cell 61: 931). The PCRproducts were then digested with ApaI (Gibco BRL, Gaithersburg, Md.,USA) for 2 h at 30 C. Digested PCR products were electrophoresed on 8%denaturing polyacrylamide gels and visualized by autoradiography. Thepresence of mt3243 led to the cleavage of the 427 bp product into 213and 214 bp fragments. Standards containing 0-100% mutant mt3243 (made bymixing a cloned DNA carrying no mt3243 mutation and another cloned DNAcarrying >99% mutant mt3243 in different proportions, kindly given by DrJ. van den Ouweland, Leiden University) were also included in the assay.The 100% mutant DNA was used as a positive control to evaluatecompleteness of PCR product digestion. The intensity of bands wasquantified by a Bio-Rad Model GS-670 imaging densitometer and aMolecular Analyst software (version 1.3) (Bio-Rad, Hercules, Calif.,USA). The proportion of mt3243 in a sample was calculated by dividingthe intensity at mutant bands (213 and 214 bp) by the total intensity ofboth wild-type and mutant bands.

Clinical Studies

All patients underwent a structured assessment including documentationof family history, age of diagnosis, body mass index (BMI) andwaist-to-hip ratio. Audiometry was performed by a technician at theotolaryngology department to assess the sensorineural status in subjectscarrying the mt3243 mutation. A fasting blood sample was taken for themeasurement of glucose, C-peptide, insulin and glycosylated haemoglobin(HbA_(1c)). Insulin resistance (IR) was estimated using the homeostasismodel assessment (HOMA) where IR=fasting insulin×fasting glucose/22.5(Matthews et al, (1985) Diabetologia 28: 412). Obesity was defined as aBMI≧27 kg/m² in men and ≧25 kg/m² in women (National Diabetes DataGroup, 1979). The basal pancreatic β-cell reserve was also assessed byplasma fasting C-peptide level. Patients with a C-peptide level≦0. 2nmol/l were considered to be insulin deficient (Service et al,. (1997)Diabetes Care 20: 198).

Biochemical Assays

Plasma glucose was measured by a glucose oxidase method (DiagnosticChemicals, Charlottetown, PEI, Canada). HbA_(1c) was measured by gelelectrophoresis (Ciba Coming Diagnostics Corp, Palo Alto, Calif. USA)C-peptide was measured by radioimmunoassay (Novo-Nordisk, Copenhagen,Denmark) with an intra-assay coefficient of variance (CV) of 3.4% andinterassay CV of 9.6%. Insulin was measured by radioimmunoassay(Pharmacia, Uppsala, Sweden) with in intra-assay CV of 6% and interassayCV of 13.8%.

Statistical Analysis

Data are expressed as mean ±SD or median (range) as appropriate. The X²test was used for analysing categorical data. Spearman correlation wasused for measurement of association between variables. All statisticswas performed with the Statistical Package for Social Sciences (SPSS)for Windows, version 6.1. A P-value<0.05 was considered as significant.

Results

The clinical details of the 906 type 2 diabetic patients are shown inTable 5. A significantly higher prevalence of maternal over paternalhistory of diabetes was found in both early- (Group 1) and late-onset(Group 3) diabetic patients with a positive family history (Table 5).

Amongst the 906 type 2 diabetic patients, in addition to the twopatients reported previously (Smith, et al (1997) supra), three morepatients carrying the mt3243 mutation were identified. In Group 1, thismutation was found in 1.8% of (four of 219) early onset patients with apositive family history. This prevalence increased to 3% (four of 133)if only those with a positive maternal family history were considered.In addition, one of the 348 late-onset patients without a family history(Group 4) was found to have this mutation (0.3%). None of the 128 earlyonset patients who had no family history (Group 2) or 211 late-onsetpatients with a positive family history (Group 3) or 213 controlsubjects had the mutation.

Amongst the five probands with the mt3243 mutation, three families wererecruited (FIG. 8). In family A, family members with diabetes or IGTwere identified but none of them carried the mutation. In Family E, twomore subjects were found to have the mutation of whom one had diabetes.The clinical and biochemical characteristics of subjects carrying themt3243 mutation are summarized in Table 6.

The percentage of mt3243 varied from 1% to 14% (Table 6). There was nocorrelation of heteroplasmy level of mutation with levels of HbA_(1c)fasting plasma glucose, C-peptide, insulin, insulin resistance or thepresence of sensorineural impairment (P>0.05) (Table 6).

Families A and B

These two families have been reported in a previous study (Smith, et al,(1997) supra). The 37-year-old proband (II 4) in family A had beentreated with oral drugs since diagnosed at the age of 32 years. Themother (I-2) and two siblings (II-1 and II-3) were diabetic while thefather (I-1) and one sister (II-2) had IGT. However, none of the familymembers had the mt3243 mutation although the mother had a history ofhearing loss.

The proband of family B was diagnosed as having diabetes at the age of22 years and had been treated with oral drugs. The mother was diabeticand deaf but was not available for screening.

Family C

The 38-year-old proband (III-3) was diagnosed with diabetes at the ageof 31 years. She was treated with diet and oral drugs for 6 years beforebeing changed to insulin therapy. Audiometry revealed bilateral hightone sensorineural impairment. The father (II-1) had normal glucosetolerance and did not have the mt3243 mutation. The older sister (III-2)and the mother (II-2) both developed diabetes at about 40 years of ageand the grandmother had diabetes at the age of 50 years. The motherbecame deaf at the age of 59 years. None of these affected members wereavailable for screening. TABLE 5 The clinical and biochemical featuresof 902 Chinese patients with type 2 diabetes Group 1 Group 2 Group 3Group 4 (n = 219) (n = 128) (n = 211) (n = 348) Age of diagnosis (year)  32 ± 6   32 ± 7   52 ± 8   57 ± 9 Duration of disease (years)   4(0-31)   2 (0-41)   5 (0-24)   4 (0-26) Sex (M/F) (%) 36/64 36/64 44/5641/59 Family history of diabetes Father (%) 45*  0 21**  0 Mother(%) 61 0 38  0 Siblings(%) 35  0 57  0 At least 1 parent and 1 sibling(%) 26 0 19  0 Body mass index (kg/m²) 25.7 ± 4.8 24.9 ± 4.4 24.4 ± 3.8 24.3 ±3.8 HbA_(lc) (%)  7.3 (4.1-15.3)  7.1 (3.8-16.8)  7.7 (4.0-16.0)  7.6(4.2-19.7) Fasting plasma glucose (nmol/l)  7.4 (4.4-23.0)  7.7(2.8-21.4)  7.8 (3.9-34.0)  8.1 (3.0-24.5) Fasting plasma C-peptide(nmol/l) 0.47 (0.03-4.96) 0.56 (0.09-1.62) 0.57 (0.01-9.40) 0.51(0.01-8.22) Insulin deficiency (%)† 16 11 12 16 Insulin treatment(%) 1113  9 11Mean ± SD or median (range). Group 1: early onset (40 years) patientswith a family history of diabetes; Group 2: early onset patients withouta family history of diabetes; Group 3: late-onset patient (>40 years)with a family history of diabetes; Group 4: late-onset patients withouta family history of diabetes.†Insulin deficiency defined as fasting plasma C-peptide 0.2 nmol/l(Service, et al (1997) supra);*P < 0.005 and**P < 0.0001 for comparison between prevalence rates of paternal vs.maternal family histories.

TABLE 6 The clinical and biochemical characteristics of Chinese subjectscarrying the mt3243 mutation in the mitochondrial tRNA^(Leu) gene FamilyA Family B Family C Family D Family E proband proband proband probandproband II-2 II-4 Age of diagnosis (yr) 32 22 31 70 33 38 — Duration ofdisease (yr) 2 1 7 9 2 1 — Sex F M F M M F F Body mass index (kg/m²)18.2 22.4 18.6 25.3 27.6 21.6 19.6  Waist-to-hip ratio 0.79 0.81 0.750.90 0.86 0.79  0.75 HbA_(1c) (%) 5 7.7 6.8 5.9 11.3 7.5 4.3 Fastingglucose(mmol/l) 5.3 11.6 9.8 5.8 15.7 7.1 4.4 Fasting C-peptide (nmol/l)0.43 0.23 0.30 0.7 0.72 0.83 0.4 Fasting insulin (mIU/I) 13.4 26.0 ND ND20.6 16.8 13.2  Insulin resistance* 3.2 13.4 5.5 ND 14.4 5.3 2.6Treatment Oral drugs Oral drugs insulin Oral drugs Oral drugs Oral drugs— Audiogram Normal Normal High tone High tone Normal ND ND impairmentImpairment Mt3243 level (%) 13 1 14 1 5 9 4*HOMA method,ND, not determinedFamily D

The 79-year-old proband was diagnosed with diabetes at the age of 70years. An audiometry test revealed high tone sensorineural impairment.Neither of the parents and nor any siblings were available for screeningor known to have diabetes.

Family E

The 35-year-old proband (II-3) was diagnosed with diabetes at the age of33 years and was treated with diet. One of the sisters (II-4) had normalglucose tolerance while two sisters (II-1 and II-2) were diagnosed tohave diabetes at the age of 30 and 38 years, respectively. The father(I-1) and mother (I-2) also had diabetes at the age of 50 and 35 years,respectively. All the diabetic and nondiabetic sisters who came forscreening had the mt3243 mutation. One of the diabetic sisters (II-1)had high tone sensorineural impairment whereas the audiogram of theproband was normal.

EXAMPLE 3 The Role of the Amylin Gene S20G Mutation in Early Onset Type2 Diabetes and in the Regulation of Cholesterol Metabolism in Chinese

This example illustrates the distribution of the amylin gene S20Gmutation in Hong Kong Chinese with or without Type 2 diabetes, and itsinfluences on β-cell function and metabolic profiles. The data areconsistent with the conclusion that the S20G mutation in the amylin genemay contribute to early occurrence of Type 2 diabetes, and that it mayalso influence lipid metabolism in the Chinese population.

Experimental Design and Methods

Subjects

The study protocol was approved by the Clinical Research EthicsCommittee of the Chinese University of Hong Kong. Informed consent wasobtained from each of the participants. For the study, 227 early-and 235late-onset Type 2 diabetic patients (defined as age at diagnosis ≦40and >40 years respectively), as well as 126 non-diabetic subjects(defined as fasting plasma glucose <6 mmol/1), were consecutivelyrecruited at the Diabetes Centre of the Prince of Wales Hospital. Type 2diabetes was asscertained according to the World Health Organisationcriteria (Anonymous (1997) Diabetes Care 20: 1183). None of the patientshad typical presentations of Type 1 diabetes, such as acute symptoms andheavy ketonuria (>3+), history of diabetic ketoacidosis or continuousneed for insulin treatment within I year of diagnosis. Patients who hadanti-glutamic acid decarboxylase autoantibody (Ko, et al (1998) Ann ClinBiochem 35: 761) diabetes-causing mutations in the glucokinase andhepatonuclear factor-1α and 4-α genes (Ng et al (1999) Diabetic Medicine16: 956) were excluded from the study.

Clinical and Biochemical Measurements

Patients fasted at least 8 hours prior to their clinical examinations.Blood pressures were taken, after they remained sitting for at least for5 min using a standard mercury sphygmomanometer. Body height and weight,and waist and hip circumferences were taken while the patients werestanding in light clothing but wearing no shoes. Measurements of fastingplasma glucose and lipids were performed by routine assays in theDepartment of Chemical Pathology at the Prince of Wales Hospital. Levelsof total cholesterol and triglyceride were assayed enzymatically withcommercial reagents(Centrichem, chemistry system, Baker Instrument Co.,Allentown, Pa.). HDL-cholesterol was determined after fractionalprecipitation with dextran sulfate-MgCl₂ and LDL-cholesterol, calculatedby the Friedewald's equation (Friedewald et al (1972) Clin Chem 18:499).HbA_(1c) was measured using an automated ion-exchange chromatographicmethod (BioRad, Hercules, Calif., USA; normal range: 5.1-6.4%). Plasmalevels of C-peptide were measured by radioimmunoassays using commercialkits (#7350104 and #141 respectively, Novo Nordisk, Denmark). Thedetection range was from 0.01 to 1.0 pmol/1. Insulin deficiency wasdefined as fasting plasma C-peptide level <0.2 pmol/1 (Service et al(1997) Diabetes Care 21: 987).

Mutation Detection

The S20G mutation creates a MSP I restriction fragment lengthpolymorphism (RFLP), which can be detected using PCR-RFLP analysis(Sakagashira et al (1996) Diabetes 45: 1279). Briefly, DNA fragmentsspanning the mutation site were amplified by PCR using the primers5′-TCACATTTGTTCCATGTTAC-3′ (SEQ ID NO: 30) and5′-CAATAACTATAGAGTTACATTG-3′ (SEQ ID NO: 31), at the annealingtemperature of 56° C. Each of the PCR products was then digestedovernight with 5 units of the restriction enzyme MSP I (#R6401, Promega,Wis., USA) at 37° C. Alleles were separated on 2.5% agarose gel. Thewild-type allele showed a 239 bp product whereas the mutant alleleshowed 99 and 140 bp products.

Statistical Analysis

Continuous variants were expressed as mean ±SD. Chi-square test was usedfor the analysis of proportions. Differences between continuousvariables were analysed by the student's t-test using the statisticalpackage for social sciences (SPSS Inc., Chicago, USA). ap value <0.05was considered to be statistically significant.

Results

Table 7 summaries the demographic data of the subjects involved in thestudy.6 early-and 1 late-onset patients heterozygous for the amylin S20Gmutation (2.6% vs 0.4%, p=0.055) were identified. None of thenon-diabetic subjects had the S20G mutation (Table 7).

In the early-onset group, 5 out of the 6 mutation-carrying patients hadsatisfactory glycemic control with diet and/or oral drug medications,and had fasting plasma C-peptide concentrations of greater than 0.2pmol/1 (Table 8). Moreover, the mutation carriers had lower totalcholesterol (4.3±0.9 vs 5.3 1.1, p=0.02) and LDL-cholesterol (2.3±0.7 vs3.2±0.9, p=0. 01) (Table 9) than those without the mutation. Thepatients with or without the S20G mutation were of a comparable age(34±6 vs 35±8, p>0.05). TABLE 7 Clinical characteristics of the earlyand late-onset patients as well as non-diabetic subjects, and thedistribution of the amylin gene S20G mutation. Type 2 diabetes Controlsubjects Early-onset Late-onset Clinical characteristics n 126 227 235Age (years) 34.9 ± 10.4 36.8 ± 6.7  59.4 ± 10.1 Sex ratio (M/F) 1:1.751:1.97 1:1.33 Age of diagnosis (years) NA 31.7 ± 4.6  54.3 ± 9.8  Bodymass index (kg/m2) 22.3 ± 3.4  25.1 ± 4.5  24.2 ± 3.6  Waist to hipratio 0.77 ± 0.05 0.85 ± 0.07 0.89 ± 0.06 Systolic blood 114 ± 10  119 ±17  137 ± 21  pressure (mmHg) Diastolic blood 64 ± 9  76 ± 10 82.0 ±11   pressure (mmHg) HbA_(lc) (%) — 7.6 ± 2.0 8.1 ± 2.2 Totalcholesterol (mmol/l) 4.7 ± 0.9 5.3 ± 1.2 5.6 ± 1.2 HDL-cholesterol(mmol/l) 1.4 ± 0.3 1.3 ± 0.4 1.3 ± 0.4 LDL-cholesterol (mmol/l) 2.9 ±0.8 3.2 ± 0.9 3.6 ± 1.1 Triglyceride (mmol/l) 0.9 ± 0.5 1.7 ± 1.8 1.8 ±1.5 Genotypes Wild-type allele 126 221 234 homozygotes Heterozygotes 0 61 Mutant allele 0 0 0 homozygote S20G allele 0 2.6^(‡) 0.4 frequency (%)Mean ± SD;‡p = 0.055

TABLE 8 Fasting plasma levels of glucose, HbA1c and C-peptide inearly-onset Type 2 diabetic patients with an amylin gene S20G mutationC- Onset Duration peptide HbA_(1c) Glucose Patient age (years) Treatment(pmol/l) (%) (mmol/l) Index a 29 1 Diet >1.0 6.2 5.3 Index b 25 18 Oraldrugs >1.0 7.2 7.2 Index c 35 3 Diet >1.0 8.2 9.3 Index d 28 2 Diet +oral 0.02 5.4 4.9 Index e 36 1 Diet + oral 0.51 5.8 14.0 Index f 13 13Insulin — 11.5 7.2

TABLE 9 Comparisons of clinical characteristics and biochemicalmeasurements between early-onset S20G mutation-carrying patients andearly-onset patients without the S20G mutation. Patients Age Sex BMI WHRSBP DBP HbA_(1c) Triglyceride Total-C HDL-C LDL-C Index a 30 F 23.2 0.76114 79 6.2 0.76 3.8 1.04 2.5 Index b 43 F 17.9 0.78 138 74 7.2 0.46 5.82.21 3.3 Index c 38 M 28.2 0.88 128 90 8.2 2.90 4.7 1.06 2.3 Index d 30F 23.2 0.75 105 66 5.4 0.99 4.0 1.37 2.2 Index e 37 F 24.0 0.89 126 805.8 2.76 3.3 0.87 1.2 Index f 26 M 29.1 0.89 120 81 11.5  0.59 4.2 1.712.2 Mutation + (n = 6) 34 ± 6 — 24 ± 4 0.8 ± 0.1 122 ± 16 78 ± 8  7.4 ±2.0 1.4 ± 1.1 4.3 ± 0.9 1.3 ± 0.5 2.3 ± 0.7 Mutation − (n = 221 35 ± 8 —25 ± 4 0.9 ± 0.1 119 ± 17 76 ± 10 7.6 ± 2.3 1.7 ± 2.5  5.3 ± 1.1* 1.3 ±0.4  3.2 ± 0.9**Mean ± SD;*p = 0.02;**p = 0.01BMI, body mass index (kg/m²);WHR, waist to hip ratio;SBP, systolic blood pressure (mmHg);DBP, diastolic blood pressure (mmHg);Total-C, total cholesterol (mmol/l);HDL-C, HDL-cholesterol (mmol/l);LDL-cholesterol (mmol/l).

The genetic association between the S20G mutation and early-onset Type 2diabetes (Table 7 ; Sakagashira et al (1996) supra) is consistent withthe physiological data that amylin may play a role in the pathogenesisof the disease (Cooper (1994) Proc Natl Acad Sci 84: 8628). Early onsetof Type 2 diabetes is in fact not uncommon (Rosenbloom et al (1999)Diabetes Care 22: 345), although type 2 diabetes is classically alate-onset disease. However, early-onset patients appear to beheterogenous in etiology. In Hong Kong Chinese, in particular,maturity-onset Type 2 diabetes of the young as well as atypicalautoimmune diabetes are present, but accounting for only a smallproportion of the overall early-onset population (Ng et al (1999) supra;Ko et al (1998) supra). The S20G mutation may also explain some of theearly-onset cases.

The S20G mutation carriers usually did not require insulin for glycemiccontrol, and did not appear to be insulin deficient (Table 8). Thesefindings are different from previous observations that the S20G mutationmay be associated with poor glycemic control as well as β celldysfunction (Sakagashira et al (1996) supra; Chuang et al (1998) supra).The reported Japanese S20G carriers (Sakagashira et al (1996) supra) hadan average diabetes duration of approximately 20 years at the time theywere tested. That they commonly required insulin treatment may be due tothe deterioration in glycemic control during their long diabetes course,not necessary the presence of the mutation.

Moreover, the mutation appears to be associated with lower plasma levelsof total cholesterol and LDL-cholesterol (Table 9). This is in keepingwith the recent finding that pramlintide (a synthetic human amylinanalog) was able to lowers plasma levels of total cholesterol andLDL-cholesterol in Type 2 diabetic patients (Thompson et al (1998)Diabetes Care 21: 987). Few studies to date have been focused on therelationships between amylin action and lipid profiles These data andthose from Thompson and co-workers are consistent with the conclusionthat amylin may also play a role in the regulation of cholesterolmetabolism.

EXAMPLE 4 The Significant Roles of Genetics and Obesity in FamilialEarly-Onset Type 2 Diabetes in Chinese Patients

This example illustrates the prevalence of known molecular defects inseparate cohorts of Chinese patients with early-and late-onset familialType 2 diabetes. The genes studied are those that have been found to beassociated with diabetes and which may contribute to early onset of thedisease under gene-gene and gene-environmental influences, includingglucokinase (MODY2), HNF-1α (MODY3), and the A3243G mutation in themitochondrial DNA coding for tRNA^(Leu(UUR)) (mt3243) that has beenassociated with a form of diabetes characterized by maternal inheritanceand deafness (van den Ouweland, et al (1992) Nature Genet. 1: 368).

Experimental Design and Methods

Subjects

The Prince of Wales Hospital (PWH) is a regional teaching hospital inHong Kong. Its catchment area has a population of 1.2 million,accounting for 20% of the total population in Hong Kong. There is a lackof long term health care programs in Hong Kong, and medical insurance isnot widely available. Many patients with chronic diseases such asdiabetes are managed in public hospitals or clinics where they pay onlya nominal fee. Hence, except for high social classes, the patients arelargely representative of the diabetic population in Hong Kong. Since1995, all patients attending the diabetes clinic of the PWH have beenentered into the PWH Diabetes Registry after undergoing a structuredassessment (Piwemetz, et al (1993) Diabetic Med 10:371; Chan, et al(1997) Hosp Auth Qual Bull 2:3). During the study period, a separatecohort of 150 young patients with early-onset diabetes (age ≦40 yearsand age at diagnosis ≦35 years) who underwent the structured assessmentwere recruited consecutively from the diabetes clinics at the PWH toform the Young Chinese Diabetes Database (Ko, et al (1998) Ann ClinBiochem 35: 761). The 150 cases in the Young Chinese Diabetes Database,92 and 53 patients, respectively, were selected for the present study asthey satisfied the following criteria: All these 145 young patients hadearly-onset (current age and age at diagnosis ≦40 years) Type 2 diabetes(1985 WHO criteria, Geneva) and a positive family history for diabetes(at least 1 first degree relative with diabetes). Patients withclassical Type 1 diabetes (acute ketotic presentation or continuousrequirement of insulin within 1 year of diagnosis) were excluded fromthe study.

The prevalence of anti-GAD (Ko, et al (1998) supra), mt3243 (Smith, etal (1997) Diabetic Med 14: 1026; Ng, et al (2000) 52: 557) and amylingene mutations (Lee, et al (2001) J. Endocrinol) amongst patients fromthe Young Chinese Diabetes Database has been reported. Additionally, theprevalence of mt3243 (Ng, et al (2000) supra), amylin (Lee, et al (2001)supra), glucokinase, HNF-1α and HNF-4α gene mutations (Ng, et al(1999)Diabetic Med 16:956) in a separate cohort from the PWH Diabetes Registryhas been reported. In this study, screening for glucokinase and HNF-1αgene mutations was extended to the 53 patients from the Young ChineseDiabetes Database. The HNF-4α gene was not screened in this cohort dueto the expected low frequency of mutations. (None were found in the 92patients from the PWH Diabetes Registry (Ng, et al (1999) supra)).Screening for anti-GAD was extended to the 92 patients from the PWHDiabetes Registry.

Nineteen (13%) of these 145 young patients with familial diabetes metthe minimal criteria for MODY (age at diagnosis ≦25 years and presenceof diabetes in two consecutive generations). Altogether 10 out of 20families with probands carrying putative diabetogenic gene mutationswere recruited for a 75-gram OGTT and clinical assessment. The 1999 WHOclassification was used to define the glycemic status of the familymembers (WHO, Geneva, 1999). For comparison of clinical characteristicsof the early-onset patients, 290 sex-matched patients with late-onsetdiabetes (age at diagnosis >40 years) and a positive family history ofdiabetes were randomly selected from the current 1800 cases in the PWHDiabetes Registry. One hundred healthy Chinese (age 33±10 years, 40males and 60 females) were selected as control subjects from hospitalstaff and students for screening for the gene variants identified in thestudy patients. Informed consent was obtained from each subject for ablood sample to be taken for DNA extraction and measurement ofbiochemical indices. This study was approved by the Clinical ResearchEthics Committee of The Chinese University of Hong Kong.

Clinical Studies

All patients underwent a structured assessment based on the EuropeDiabCare Protocol. They had documentation of their family history ofdiabetes, age at diagnosis and anthropometric indices (Piwemetz, et al(1993) supra; Chan, et al (1997) supra). Body mass index (BMI) was usedas an index of general obesity. Waist circumference, which is highlycorrelated in Chinese with visceral fat accumulation measured bymagnetic resonance imaging (Anderson, et al (1997) Diabetes Care 20:1854), was used as an index of central obesity. After an overnight fast,venous blood was sampled for measurement of plasma glucose, insulin,HbA_(1c), total cholesterol (TC), HDL-C, LDL-C (calculated),triglyceride (TG) and anti-GAD. A morning spot urine sample wascollected for assessment of albuminuria. Retinopathy and sensoryneuropathy were assessed as previously described (Ko, et al (1999) JDiabetes Complications 13: 300).

General obesity was defined as a BMI≧25 kg/m² using the recent Asiancriteria (WHO, Western Pacific Region, 2000). Albuminuria was defined asan albumin: creatinine ratio (ACR)≧3.5 mg/mmol in a spot urine sample(Schwab, et al (1992) Diabetes Care 15: 1581). The HOMA IR index(fasting plasma insulin×glucose/22.5) derived from the HOMA equation wasused to assess insulin resistance (Matthews, et al (1985) Diabetologia28: 412).

Biochemical Assays

Plasma glucose, HbA_(1c), lipids, urinary albumin and creatinine weremeasured by routine assays in the Department of Chemical Pathology atthe PWH (see Chan, et al (1996) Diabetic Med 13:150). Plasma insulin wasmeasured in non-insulin treated patients by a radioimmunoassay(Pharmacia, Sweden) with intra-and inter-assay CVs of 6% and 13.8%,respectively. Anti-GAD was measured by a radioimmunoprecipitation assay(Chen, et al (1993) Pediatr Res 34: 785). The upper normal limit of 18units, is applicable to Asian and European subjects (Tuomi, et al (1995)Clin Immunol Immunopath 74: 202, Chen, et al (1993) supra).

Genetic Analysis

The minimal promoter regions and exons of the glucokinase (β-cell form),HNF-1α and HNF-4α (HNF-4α 2 form) genes were screened for mutations bydirect sequencing of PCR products (see Froguel, et al (1993) N Engl JMed 328: 697; Yamagata, et al (1996) Nature 384: 455; Yamagata, et al(1996) Nature 384: 458). One previously unreported mutation in HNF-1α(A116V) and two previously unreported mutations in glucokinase (V101Mand Q239R) were identified in this study. HNF-1α A116V was screened byusing the forward primer 5′-CATGCACAGTCCCCACCCTCA-3′ (SEQ ID NO:34) andreverse primer 5′-TCCCACTG ACTTCCTTTCC-3′ (SEQ ID NO:35) for PCRamplification followed by digestion with HphI. The wild-type alleleshowed 44 and 397 bp products whereas the mutant allele showed 44, 136and 261 bp products. Glucokinase V101M was screened by using the forwardprimer 5′-GTCCCTGA-GGCTGACACACTT-3′ (SEQ ID NO: 24) and reverse primer5′-AGCTGGGCCCTGAGATCC-TGCA-3′ (SEQ ID NO: 25) for PCR amplificationfollowed by digestion with Hsp9211. The wild-type allele showed 20, 56and 174 bp products whereas the mutant allele showed 20,42,56, and 132bp products. Glucokinase Q239R was screened by using the forward primer5′-AGGAACC-AGGCCCTACTCCG-3′ (SEQ ID NO: 36) and reverse primer5′-TACTCCAGCAGGAACTC-GTCC-3′ (SEQ ID NO: 37) for PCR amplificationfollowed by digestion with Acil. The wild-type allele showed 70 and 134bp products whereas the mutant allele showed 33,70 and 101 bp products.The occurrence of putative mutations in family members of the probands(Table 10) and control subjects was determined by PCR-RFLP. Mt3243A→Gand amylin gene S20G mutations were determined by PCR-RFLP as described(Sakagashira, et al (1996) Diabetes 45: 1279; Smith, et al (1997)supra).

Statistical Analysis

Normally distributed data are expressed as mean ± SD. Data with skeweddistributions were normalised by logarithmic transformation. Theresultant means were antilogarithmically transformed and expressed asgeometric mean together with 25 and 75 percentiles. Chi square test andStudent's unpaired t tests were used for between-group comparisons. A pvalue<0.05 (2-tailed) was considered to be significant. All statisticalanalyses were performed using the Statistical Package for SocialSciences (SPSS for Windows, version 9.0).

Results

Prevalence of Putative Gene Mutations and Anti-Gad in Patients withFamilial Early-Onset Diabetes

Amongst the 145 patients with familial early-onset diabetes, there were20 (14%) with putative mutations, involving the HNF-1α gene, 7 (5%),glucokinase gene, 6 (4%), mt3243,4 (3%), and amylin S20G, 3 (2%).Anti-GAD was positive in 6 (4%). No mutation in the HNF-4α gene wasfound in the 92 patients from the PWH Diabetes Registry. All mutationsidentified in the HNF-1α and glucokinase genes were previouslyunreported (Table 10). The HNF-1α G20R and glucokinase Q239R mutationswere found in 4 unrelated patients. None of these mutations were foundin 100 healthy control subjects.

Family Cosegregation Study of Gene Mutations

Amongst the 20 patients carrying putative gene mutations, 10 familieswere recruited for cosegregation study (FIG. 9). Cosegregation of amutation with clinical diabetes or glucose intolerance were observed in4 families: HK10 with HNF-1α IVS2nt-1G→A, YDM142 with glucokinase V101M,HK84 with glucokinase I110T and HK50 with mt3243. Segregation wasinconclusive in the other 6 families. For the families HK54 with HNF-1αR203H, YDM83 with mt3243 and CX216 with amylin S20G mutations, only theprobands, and none of the diabetic or non-diabetic family members whopresented for screening, carried the gene mutations. For YDM67 withglucokinase Q239R, HK61 with mt3243 and YDM99 with amylin S20Gmutations, the mutations were found in both diabetic and non-diabeticfamily members. Amongst the 3 families with glucokinase mutations, allmutation carriers from the families YDM142 and HK84 had higher fastingplasma glucose concentrations (5.8-8.9 mmol/1) than those with nomutation (4.2-5.3 mmol/1). On the other hand, the 4 mutation-carryingsiblings of proband YDM67 had a normal fasting plasma glucoseconcentration (4.0-5.6 mmol/1) irrespective of their glycemic status.TABLE 10 Mutations in the HNF-1 α and glucokinase genes in Chinesesubjects with early-onset diabetes mellitus Subject Location Codon/ntNucleotide change Designation HNF-1 _(α)mutation HK90*, YDM42† Exon 1 20 GGG (GLy)→AGG (Arg) G20R YDM20† Exon 2 116 GCG (Ala)→GTG(Val) A116VHK10* Intron2/Exon3 nt-1 AG→AA at splice acceptor site IVS2nt-1G→A HK54*Exon 3 203 CGT (Arg)→CAT (His) R203H HK30* Exon 6 432 TCC (Ser) →TGC(Cys) S432C HK92* Exon 10 618 ATC (Ile)→ATG (Met) 1618M Glucokinasemutation YDM142† Exon 3 101 GTG (Val)→ATG (Met) V101M HK84* Exon 3 110ATC (Ile)→ACC (Thr) I110T HK38* Exon 3 119 GCT (Ala)→GAT (Asp) A119DYDM67†, YDM144† Exon 7 239 CAG (Gln)→CGG (Arg) Q239R HK15* Exon 9 385GGG (Gly)→GTG(Val) G385V*reported in previous studies (Ng, et al (1999) Diabetic Med 16: 956;Ng, et al (2000) Diabetologia 43: 816)†newly found in the present studyClinical Characteristics of Patients with Familial Early-Onset Diabetesof Unknown Cause Compared with Familial Late-Onset Diabetes

Although 26 of the patients with early-onset diabetes carried putativegene mutations associated with diabetes or the autoimmune indicator,anti-GAD antibodies, the causes of diabetes in the other 119patientsremain to be determined. These young patients with diabetes ofunknown cause (age at diagnosis 30±6 years) differed clinically from the290 late-onset patients (age at diagnosis 52±8 years) (Table 11). Thus,despite a positive family history of diabetes in all patients in bothgroups, those with early-onset diabetes more frequently had a fatherwith diabetes (39% vs. 22%) and a mother with diabetes (63% vs. 41%),but less frequently a sibling with diabetes (30% vs. 53%) (p<0.001). Theearly-onset patients had a higher BMI but lower BP and increasedprevalence of retinopathy and neuropathy as compared to the late-onsetpatients. The early-onset patients had better glycemic control (glucoseand HbA_(1c)) as well as higher fasting insulin concentrations than thelate-onset patients. Notwithstanding similar mean disease duration ofonly 4 years, both the early-and late-onset patients had adisproportionately high prevalence of albuminuria, 40% and 38%,respectively, as compared with the prevalence rates of othermicroangiopathic complications. Insulin resistance, as assessed by theHOMA IR index, was similar between the two groups of non-insulin treatedpatients. The proportion of patients treated with insulin was similar inboth groups (8% vs. 7%) but fewer patients with early-onset diabeteswere treated with oral drugs (33% vs. 61%, p<0.001) as compared to thelate-onset group.

Clinical Characteristics of the Patients with Familial Early-OnsetDiabetes of Unknown cause and Familial Late-Onset Diabetes ClassifiedAccording to Obesity Index

Due to the high prevalence of general obesity in both early-onsetpatients of unknown cause and late-onset patients (55% and 46%,respectively), the association of obesity with cardiovascular riskfactors and complications in these patients was further analyzed (Table11). Amongst the early-onset patients, the obese patients had worseglycemic control (HbA_(1c)) as well as a higher systolic BP, a moreadverse lipid profile (higher TG, lower HDL-C and higher TC/HDL-C), andhigher fasting insulin than the non-obese patients. They were also moreinsulin resistant (HOMA IR index) and had a higher prevalence ofretinopathy and albuminuria than the non-obese patients. Amongst thelate-onset patients, the obese patients had better glycemic control(glucose and HbA_(1c)) than the non-obese patients. However, they had ahigher systolic and diastolic BP, and a higher fasting insulin than thenon-obese patients. The degree of insulin resistance and prevalence ofcomplications were similar in the two groups. TABLE 11 Comparison ofclinical features of Chinese patients with familial Type 2 diabetesaccording to age of diagnosis of diabetes and obesity status Early-onsetLate-onset Early-onset patients Late-onset non-obese Early-onsetnon-obese Late-onset with unknown etiology patients patients obesepatients patients obese patients   N  119  290   54   65  156  134 SexMale (%)   37 (31)   98 (34)   12 (22)   25 (38)   56 (36)   42 (31)Female (%)   82 (69)  192 (66)   42 (78)   40 (62)  100 (64)   92 (69)Current age (yr)   34 ± 5   56 ± 9‡   34 ± 5   33 ± 5   56 ± 10   55 ± 9Age at diagnosis (yr)   30 ± 6   52 ± 8‡   31 ± 5   29 ± 6   52 ± 8   52± 8 Duration of disease (yr)  4.0 ± 3.9  4.0 ± 4.2  3.9 ± 3.8  4.2 ± 4.0 4.5 ± 4.4  3.5 ± 3.9† Family history Father   46 (39)   64 (22)‡   21(39)   27 (42)   28 (18)   36 (27) Mother   75 (63)  119 (41)‡   38 (70)  39 (60)   65 (42)   54 (40) Sibling   36 (30)  154 (53)‡   15 (28)  20 (31)   85 (54)   69 (51) BMI (kg/m²) 26.2 ± 4.7 25.0 ± 3.7† 22.3 ˜1.8 29.5 ± 3.8‡ 22.4 ± 1.8 28.0 ± 3.1‡ Waist circumference (cm) Male  90 ± 11   87 ± 9   78 ± 6   95 ± 9‡   81 ± 6   94 ± 7‡ Female   81 ±11   83 ± 9   74 ± 5   89 ± 10‡   78 ± 6   89 ± 8‡ Systolic BP (mmHg) 117 ± 14  136 ± 22‡  114 ± 13  120 ± 14†  134 ± 23  139 ˜ 21† DiastolicBP (mmHg)   75 ± 9   83 ± 11‡   74 ± 9   77 ± 10   80 ± 11   86 ± 12‡Triglyceride (mmol/l)  1.4 (0.9-2.0)  1.4 (1.0-2.0)  1.0 (0.7-1.5)  1.7(1.0-2.4)‡  1.4 (0.9-1.9)  1.6 (1.1-2.1) Total cholesterol (mmol/l)  5.3± 1.2  5.6 ± 1.3  5.1 ± 1.0  5.4 ± 1.4  5.6 ± 1.3  5.5 ± 1.2 HDL-C(mmol/l)  1.2 ± 0.3  1.2 ± 0.3  1.3 ± 0.3  1.1 ± 0.3‡  1.3 ± 0.4  1.2 ±0.3 TC/HDL-C  4.7 ± 1.8  4.7 ± 1.5  4.0 ± 1.0  5.3 ± 2.2‡  4.7 ± 1.7 4.7 ± 1.3 LDL-C (mmol/l)  3.3 ± 0.9  3.5 ± 1.0  3.2 ± 0.8  3.4 ± 1.0 3.5 ± 1.0  3.5 ± 1.0 Fasting glucose (mmol/l)  8.2 ± 3.1  9.1 ± 3.6† 7.6 ± 2.8  8.7 ± 3.3  9.6 ± 3.8  8.6 ± 3.3† HbA_(1c) (%)  7.5 ± 1.8 8.0 ± 1.9†  7.1 ± 1.8  7.9 ± 1.8†  8.2 ± 2.1  7.7 ± 1.5† Fastinginsulin (pmol/l)*  105 (72-164)   87 (51-146)†   89 (60-149)  120(78-179)†   76 (43-129)   99 (57-157)† HOMA IR index* 34.7 (22.9-55.8)33.1 (19.2-62.8) 27.5 (16.0-46.7) 42.7 (27.6-58.2)† 29.9 (14.7-61.9)36.4 (19.7-64.2) Urinary albumin creatinine  2.6 (0.7-6.1)  2.8(0.8-7.1)  1.2 (0.6-1.9)  5.1 (0.9-24.1)‡  2.6 (0.9-5.8)  3.2 (0.8-8.4)ratio (mg/mmol) Treatment (%) Diet   71 (60)   93 (32)   35 (65)   36(55)   55 (35)   38 (28) Oral drugs   39 (33)  176 (61)‡   17 (31)   22(34)   89 (57)   87 (65) Insulin   9 (8)   21 (7)   2 (4)   7 (11)   12(8)   9 (7) Retinopathy (%)   10 (8)   62 (21)†   1 (2)   9 (14)†   38(24)   24 (18) Albuminuria (%)   48 (40)  110 (38)   8 (15)   40 (62)‡  53 (34)   57 (43) Neuropathy (%)   4 (3)   29 (10)†   2 (4)   2 (3)  13 (8)   16 (12)Data are compared between early- and late-onset patients, betweenearly-onset non-obese and obese patients, and between late-onsetnon-obese and obese patientsData are expressed as n (%), mean ± SD or geometric mean (25 and 75percentiles)*only measured in patients not treated with insulin†p < 0.05‡p < 0.001

EXAMPLE 5 An Illustration of a Chinese Family with Hepatocyte NuclearFactor-1α Diabetes (MODY3) that Emphasizes the Need for Early Diagnosisand Appropriate Treatment

This example reports the clinical course of HNF-1α diabetes/MODY 3 in aChinese family with early-onset diabetes and severe complications (FIG.10) (Chan, et al (1990) Diabetic Medicine 7: 211). This familyhighlights the importance of early diagnosis and prompt treatment in theimprovement of clinical outcome even in genetically susceptiblesubjects.

Three family members in the proband's family had severe diabeticcomplications when they were referred for treatment. The proband(III-5), 19 years of age, had severe proliferative retinopathy, heavyproteinuria (1.4 g protein a day) and necrobiosis lipoidica. She hadbeen diagnosed with Type 2 (non-insulin-dependent) diabetes mellitus 3months earlier and was treated with glibenclamide. Retinalphotocoagulation treatment was initiated and she was started on insulinand an ACE inhibitor. She subsequently developed hypertension andprogressed to end-stage renal disease requiring dialysis by the age of30 years. Her mean HbA_(1c) was 8.0% over the years. She is currentlyreceiving 42 units of insulin.

Her older sister (III-2) had a vitreous haemorrhage and had been treatedwith insulin since diagnosis at the age of 24 years. She became blindand had nephropathy (0.8 g protein a day) 2 years later. She iscurrently treated with insulin (16 units) and an ACE inhibitor, and hasa mean HbA , of 6.4%.

The subject's mother (II-3) had a glycosuria complicated pregnancy whenshe was 33 years old. She was diagnosed to have Type 2 diabetes at theage of 38 years and was then treated with glibenclamide for 10 years. Atthe time of the study she had proliferative retinopathy, nephropathy,peripheral neuropathy, necrobiosis lipoidica, hypertension andcataracts. Insulin treatment (20 units) was commenced and her HbA_(1c)was reduced from 17.2% to 9.2% within 8 months. Two months later, shehad a myocardial infarction followed by progressive deterioration ofcardiac and renal functions. She died of pulmonary edema and septicaemiawith a gangrenous foot at the age of 52 years.

The fourth daughter (III-6) had been treated with insulin since herincidental diagnosis of diabetes at the age of 12 years after a nasalpolypectomy. She is currently receiving 68 units of insulin, and hasmean HbA_(1c) of 8.8%.

Two other family members underwent screening by OGTT. The seconddaughter (III-3) has fluctuated between having normal glucose toleranceand IGT over the last 11 years. A brother (III-7) had overt diabetes onscreening with an initial HbA_(1c) of 10.5%. Insulin was started after 3months of dietary treatment. He is currently receiving 26 units ofinsulin, with a mean HbA_(1c) of 5.3%.

One maternal uncle (II-4) was diagnosed with diabetes andhyperlipidnemia with thirst and polyuria at the age of 39 years. He hasbeen treated with oral drugs since diagnosis, and has mean HbA_(1c) of8.4%. His children were not available for detailed genetic testing andclinical assessment. The affected members II-4, III-6 and III-7 (FIG.10) have remained free of complications despite all having had diabetesfor more than 10 years.

The father was also diagnosed with IGT. He was non-obese and hadhyperlipidaemia.

Sequencing of the HNF-1_(α) gene in this family showed a novel spliceacceptor site mutation (AG→AA) in intron 2 (IVS2nt-1G→A) whichcosegregated with diabetes (FIG. 10) (Ng, et al (1999) Diabetic Medicine16: 956). This mutation is expected to produce a nonfunctional mRNA. Allthe diabetic members, including the maternal uncle, (II4, III-2, III-5,III-6 and III-7) were heterozygous for this mutation but the father(II-2) and the daughter (III-3) with IGT did not have the mutation. Thusit is very likely that the mother (II-3) for whom no DNA sample wasavailable also carried this mutation. As with other patients with HNF-1αdiabetes (Byrne, et al (1996) Diabetes 45: 1503), most affected familymembers exhibited defective pancreatic beta-cell function as assessed bythe glucagon stimulation test. The mother and all the affected siblings,except subject III-2, were insulin deficient based on a definition ofpost-glucagon (1 mg intravenously) stimulated plasma C peptide at 6 minof less than 0.6 nmol/1 (0.24-0.55 nmol/I respectively) (Service, et al(1997) Diabetes Care 20: 198). The brother, III-7, who was diagnosedwith diabetes by OGTT was also insulin deficient. All the HNF-1α mutantcarriers, except II-4, required insulin treatment for glycemic control.

Although all affected family members carried the same HNF-1α genemutation, their clinical courses have varied tremendously. Severecomplications were present in those family members whose diagnosis wasdelayed and who presumably had poor glycemic control before diagnosis(II-3, III-2 and III-5). Complications were, however, absent in theuncle (II-4) and the younger siblings (III-6 and III-7) despite nowhaving had diabetes for more than 10 years (FIG. 10), who were promptlydiagnosed and received treatment. This is in accordance with a recentreport suggesting that poor glycemic control is associated with atwofold to threefold increased risk among MODY3 patients of developingmicroalbuminuria and retinopathy, respectively (Isomaa, et al (1998)Diabetologia 41: 467).

It is noteworthy that both maternal grandparents (I-3 and I-4) werediagnosed with diabetes diagnosed in their late 50 s. The effect of thisbilineality on the natural course of HNF-1α diabetes in this family isuncertain. It is, however, possible the non-MODY maternal grandparenttransmitted a modifier gene affecting the age at onset or severity ofthe diabetes in carriers with the HNF-1_(α) amutation. The age atdiagnosis of diabetes in this family was increasingly younger withsuccessive generations despite all carriers being relatively non-obese.This earlier diagnosis could be due to ascertainment bias or, morelikely, an epiphenomenon due to increasing westernisation of the HongKong lifestyle with increased intake of high fat food and decreasedphysical activity (Chan and Cockram (1997) Diabetes Care 20:1785). Thishighlights the important influence of environment interacting withgenetics in the natural course of HNF-1α diabetes. In conclusion, thisreport emphasizes the need for early diagnosis by glucose tolerancetesting or genetic screening, and appropriate treatment in patients whohave a strong family history of diabetes, especially those with earlyonset disease and insulin deficiency.

The data presented in Examples 1-5 demonstrate a combination of geneticmutations that are uniquely associated with the increased risk of aChinese individual to develop type 2 diabetes. The mutations areexemplified by, but are not limited to G20R, A116V, IVS2nt→GA, R203H,S432C and I618M of HNF-1α; V101M, I110T, A119D, Q239R and G385V ofglucokinase; S20G of amylin; and A3243G of mitochondrialtRNA^(Leu(UUR)). Mutations correlative with a genetic predisposition ofa Chinese individual to develop type 2 diabetes are efficientlyidentified in Chinese families with a positive family history of thedisease, but find use in screening any Chinese individual that isasymptomatic but at risk of developing diabetes. Methods foridentification of a combination of at least two genetic mutationscorrelative with type 2 diabetes in a Chinese individual offers animportant tool for clinicians, not only to initiate prophylactictherapies before the onset of overt diabetic symptoms, but also todesign therapies that are directed to the specific etiology of thedisease in each individual.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporate by reference.

1. A microchip comprising: a combination of at least two differentmutant nucleic acid sequences of a wild-type nucleic acid sequence,wherein each wild-type nucleic acid sequence encodes a protein involvedin insulin secretion, wherein said gene comprises at least one mutationindicative of a predisposition for type 2 diabetes in a member of aChinese population.
 2. The microchip according to claim 1, wherein saidnucleic acid sequences comprise nucleic acid selected from the groupconsisting of genomic DNA, complementary DNA and messenger RNA.
 3. Themicrochip according to claim 1, wherein said type 2 diabetes is maturityonset diabetes of the young.
 4. The microchip according to claim 1,wherein said microchip further comprises a genetic marker that uniquelyidentifies a member of a Chinese population.
 5. A microchip comprising:a combination of at least two different nucleic acid sequences, whereineach nucleic acid sequence encodes a gene product involved in insulinsecretion wherein said gene comprises at least one mutation indicativeof a predisposition for type 2 diabetes in a human subject of a Chinesepopulation, wherein said gene product is selected from the groupconsisting of a glucokinase, a hepatocyte nuclear factor 1α, an amylinand a mitochondrial tRNA (Leu) (UUR).
 6. A microchip comprising: atleast one each of a combination of different nucleic acid sequences,wherein each nucleic acid sequence encodes a protein selected from thegroup consisting of glucokinase, hepatocyte nuclear factor la, amylinand mitochondrial tRNA(Leu)(UTR), wherein said glucokinase genecomprises at least one mutation selected from the group consisting ofV101M, I110T, A119D, Q239R, and G385V, and said hepatocyte nuclearfactor la gene comprises at least one mutation selected from the groupconsisting of G20R, A116V, IVS2nt-G→A, R203H, S432C, and I618M, and saidamylin gene comprises the mutation S20G, and said mitochondrialtRNA(Leu)(UUR) gene comprises the mutation A3243G.
 7. A microchipcomprising at least one nucleic acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:10.
 8. A microassaysystem comprising a microchip according to claim
 1. 9. A kit comprisinga microchip according to claim
 1. 10. A nucleic acid primer comprised ofSEQ ID NO:
 34. 11. A nucleic acid primer comprised of SEQ ID NO:
 35. 12.A nucleic acid primer comprised of SEQ ID NO:
 36. 13. A nucleic acidprimer comprised of SEQ ID NO:
 37. 14. A nucleic acid probe thatspecifically anneals to a nucleic acid encoding a mutant gene of awild-type gene involved in insulin secretion, wherein said mutant genecomprises at least one mutation indicative of increased risk for type 2diabetes in a human subject of a Chinese population, and wherein saidnucleic acid probe does not bind to said wild-type gene.
 15. An isolatednucleic acid encoding a mutant gene of a wild-type gene that encodes aprotein involved in the secretion of insulin, wherein said mutant genecomprises at least one mutation associated with increased risk for type2 diabetes in a subject of a Chinese population.
 16. The isolatednucleic acid according to claim 15, wherein said mutation is a singlenucleotide polymorphism.
 17. The isolated nucleic acid according toclaim 15, wherein said mutation is selected from the group consisting ofa missense, a nonsense, an insertion and a deletion mutation.
 18. Theisolated nucleic acid according to claim 15, wherein said wild-type geneencodes hepatocyte nuclear factor la, and said mutation is A116V. 19.The isolated nucleic acid according to claim 15, wherein said wild-typegene encodes glucokinase, and said mutation is selected from the groupconsisting of V101M and Q239R.
 20. An isolated nucleic acid encoding amutant gene of a wild-type gene that encodes a protein involved in thesecretion of insulin, wherein said mutant gene is selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO:
 10. 21. Anisolated amino acid sequence encoded by a mutant gene of a wild-typegene encoding a protein involved in the secretion of insulin, whereinsaid mutant gene comprises at least on mutation associated withincreased risk for type 2 diabetes in a member of a Chinese population.22. An antibody that specifically binds a protein encoded by a mutantgene of a wild type gene encoding a protein involved in the secretion ofinsulin, wherein said mutant gene comprises at least on mutationassociated with increased risk for type 2 diabetes in a member of aChinese population, and wherein said antibody does not bind to a proteinencoded by said wild-type gene.
 23. A method of determining a geneticpredisposition of a member of a Chinese population to develop type 2diabetes, said method comprising the step of: contacting a samplecomprising nucleic acid from said member with a combination of at leasttwo nucleic acid sequences, wherein each nucleic acid sequence encodes amutant gene of a wild-type gene encoding a protein involved in insulinsecretion, wherein each mutant gene comprises at least one mutationindicative of a predisposition of a member of a Chinese population todevelop type 2 diabetes, whereby identification of at least one of saidmutations in said sample is indicative of a genetic predisposition fortype 2 diabetes in said member of a Chinese population.
 24. A method fordetecting an increased risk of an individual of a Chinese populationwith decreased insulin secretory function to develop type 2 diabetes,said method comprising the step of: contacting a sample comprisingnucleic acid from said individual with a combination of at least twodifferent nucleic acid sequences, wherein each nucleic acid sequenceencodes a mutant gene of a wild-type gene encoding a protein involved ininsulin secretion, wherein each mutant gene comprises at least onemutation indicative of a predisposition of a member of a Chinesepopulation to develop type 2 diabetes, wherein identification of atleast one of said mutations in said sample is indicative of an increasedrisk for type 2 diabetes in said individual of a Chinese population. 25.The method according to claim 23, wherein said combination of at leasttwo different nucleic acid sequences are attached to a microchip. 26.The method according to claim 23, wherein said nucleic acid sample isobtained from bodily fluid or tissue.
 27. The method according to claim23, wherein said wild-type gene encodes a gene product selected from thegroup consisting of hepatocyte nuclear factor 1α, glucokinase, amylinand mitochondrial tRNA(Leu)(UUR).
 28. A method of determining a geneticpredisposition of a member of a Chinese population to develop type 2diabetes, said method comprising the step of: contacting a samplecomprising nucleic acid from said member with a combination of at leasttwo different nucleic acid sequences selected from the group consistingof G20R, A116V, IVS2nt-G→A, R203H, S432C, and I618M of hepatocytenuclear factor 1α; V101M, I110T, A119D, Q239R, and G385V of glucokinase;S20G of amylin, and A3243G of mitochondrial tRNA(Leu)(UUR), wherein eachnucleic acid sequence encodes a mutant gene of a wild-type gene encodinga protein involved in insulin secretion, wherein each mutant genecomprises at least one mutation indicative of a predisposition of amember of a Chinese population to develop type 2 diabetes, and whereinsaid identification of one of said mutations in said sample isindicative of a genetic predisposition for type 2 diabetes in saidmember of a Chinese population.
 29. A method for detecting an increasedrisk of an individual of a Chinese population with decreased insulinsecretory function to develop type 2 diabetes, said method comprisingthe step of: contacting a sample from said individual with a combinationof at least two different nucleic acid sequences selected from the groupconsisting of G20R, A116V, IVS2nt-G→A, R203H, S432C, and I618M ofhepatocyte nuclear factor 1α;V101M, I110T, A119D, Q239R, and G385V ofglucokinase; S20G of amylin, and A3243G of mitochondrial tRNA(Leu)(UUR),wherein each nucleic acid sequence encodes a mutant gene of a wild-typegene encoding a protein involved in insulin secretion, wherein eachmutant gene comprises at least one mutation indicative of apredisposition of an individual of a Chinese population to develop type2 diabetes, and wherein the identification of at least one of saidmutations in said sample is indicative of an increased risk for type 2diabetes in said individual of a Chinese population.
 30. A method forscreening for genetic mutations in an individual of a Chinese populationdiagnosed with type 2 diabetes, said method comprising the steps of:contacting a sample from said individual with a combination of at leasttwo different nucleic acid sequences, wherein each nucleic acid sequenceencodes a mutant gene of a wild-type gene encoding a protein involved ininsulin secretion, wherein each mutant gene comprises at least onemutation indicative of a predisposition of a member of a Chinesepopulation to develop type 2 diabetes, and wherein identification of atleast one of said mutations in said sample is indicative of an etiologyof said type 2 diabetes in said individual of a Chinese population. 31.The method according to claim 30, wherein said individual has beendiagnosed with maturity onset diabetes of the young.
 32. The methodaccording to claim 30, wherein said individual has at least one primaryfamily member that has been diagnosed with maturity onset diabetes ofthe young.
 33. The method according to claim 30, wherein said mutationis selected from the group consisting of a missense, a nonsense, aninsertion and a deletion mutation.
 34. A method for screening forgenetic mutations indicative of increased risk of an individual of aChinese population to develop type 2 diabetes, said method comprisingthe steps of: contacting a sample from said individual with acombination of at least two different nucleic acid sequences selectedfrom the group consisting of G20R, A116V, IVS2nt-G→A, R203H, S432C, andI618M of hepatocyte nuclear factor 1α;V101M, I110T, A119D, Q239R, andG385V of glucokinase; S20G of amylin, and A3243G of mitochondrialtRNA(Leu)(UTR), wherein each nucleic acid sequence encodes a mutant geneof a wild-type gene encoding a protein involved in insulin secretion,wherein each mutant gene comprises at least one mutation indicative of apredisposition of an individual of a Chinese population to develop type2 diabetes.
 35. A method for screening for a genetic predisposition todevelop type 2 diabetes in an individual of a Chinese population havingat least one primary family member that has been diagnosed with type 2diabetes, said method comprising the steps of: contacting a samplecomprising nucleic acid from said individual with a combination of atleast two different nucleic acid sequences, wherein each nucleic acidsequence encodes a mutant gene of a wild-type gene encoding a proteininvolved in insulin secretion, wherein each mutant gene comprises atleast one mutation indicative of a predisposition of a member of aChinese population to develop type 2 diabetes, and whereinidentification of at least one of said mutations in said sample isindicative of a genetic predisposition to develop type 2 diabetes insaid individual of a Chinese population.